Devices for separation of biological materials

ABSTRACT

The present invention includes methods, devices and systems for isolating nanoparticulates, including nucleic acids, from biological samples. In various aspects, the methods, devices and systems may allow for a rapid procedure that requires a minimal amount of material and/or results in high purity isolation of biological components from complex fluids such as blood or environmental samples.

CROSS-REFERENCE

This application claims priority to U.S. Provisional Application Ser.No. 61/977,006, filed Apr. 8, 2014, and U.S. Provisional ApplicationSer. No. 61/977,249, filed Apr. 9, 2014, which is incorporated herein byreference.

BACKGROUND OF THE INVENTION

Separation of nanoscale analytes from other material present inbiological samples is an important step in the purification ofbiological analyte material, including nucleic acids, for laterdiagnostic or biological characterization. Current techniques aretypically bulky, requiring large volumes of sample for operation. Therecontinues to be a need for a robust platform capable of isolatingnanoscale analytes from complex biological samples using minimal samplevolume without requiring additional purification steps.

SUMMARY OF THE INVENTION

In some instances, the present invention fulfills a need for improvedmethods of separating nanoscale analytes from complex biological samplesutilizing minimal volumes of samples in an efficient manner. In someaspects provided herein, samples are processed and nanoscale analytesisolated in a short period of time. In other aspects, the isolatednanoscale analytes require no further sample preparation or enrichment.In still other aspects, minimal amounts of starting material is used toisolate sufficient nano scale analyte material to a desired level ofpurity and concentration such that additional analysis andcharacterization can take place without further processing orpurification. In yet other aspects, the methods, devices andcompositions disclosure herein are amenable to multiplexed andhigh-throughput operation. The nanoscale analytes isolated using themethods and devices disclosed herein are elutable and directlytransferable and capable of analysis and characterization withoutfurther manipulation to be used in other devices and methods employedfor diagnostic purposes.

In one aspect, disclosed herein, in some embodiments, are compositions,devices and methods for isolating a nanoscale analyte from a biologicalsample using a plurality of alternating current (AC) electrodes asdisclosed herein. In some embodiments, the AC electrodes are configuredto be selectively energized to establish AC electrokinetic high fields.In other embodiments, the AC electrodes are configured to be selectivelyenergized to establish AC electrokinetic low fields. In yet otherembodiments, the AC electrodes are configured to be selectivelyenergized to establish AC electrokinetic high field regions and ACelectrokinetic low field regions.

In some embodiments, the methods, devices and compositions disclosedherein utilize an array of electrode configurations and designs toimprove capture of nanoscale analytes at the surface of the electrodes.In some embodiments, the array of electrodes are configured such thatfluid flow around or within the vicinity of the electrodes are disruptedor altered, allowing the localization and/or retention of nanoscaleanalytes around or within the electrode arrays.

In some embodiments, flow around or within the vicinity of theelectrodes is substantially reduced or lessened as compared toconventional electrodes. In yet other embodiments, the reduction of flowis due to the composition of the electrode and/or electrode array. Instill other embodiments, the reduction of flow is due to the physicaldesign or configuration of the electrode and/or array. In otherembodiments, the reduction of flow is due to a combination of thecomposition of the electrode and/or electrode array as well as aphysical change in the design or configuration of the electrode and/orelectrode array. In still other embodiments, the reduction of flow isdue to compositions and/or physical configurations directly outside ofthe physical boundary of the electrode array. In yet other embodiments,the reduction of flow is due to a combination of compositions and/oralterations of physical designs and configurations of the electrodeand/or electrode array in combination with compositions and/or physicalconfigurations outside of the physical boundary of the electrode and/orelectrode array.

In some embodiments, the electrodes are capable of sourcing greater than50 mA of current. In some embodiments, the electrodes are capable ofsourcing greater than 100 mA of current. In some embodiments, theelectrodes are capable of sourcing greater than 250 mA of current. Insome embodiments, the electrodes are capable of sourcing greater than500 mA of current.

In some embodiments, disclosed herein is a device for isolating ananoscale analyte in a sample, the device comprising: (1) a housing; (2)a heater and/or a reservoir comprising a protein degradation agent; and(3) a plurality of alternating current (AC) electrodes as disclosedherein within the housing, the AC electrodes configured to beselectively energized to establish AC electrokinetic high field and ACelectrokinetic low field regions, wherein the electrodes compriseconductive material configured on or around the electrodes whichreduces, disrupts or alters fluid flow around or within the vicinity ofthe electrodes as compared to fluid flow in regions between orsubstantially beyond the electrode vicinity. In some embodiments, theconductive material is substantially absent from the center of theindividual electrodes in the array. In some embodiments, the conductivematerial is present at the edges of the individual electrodes in theelectrode array. In some embodiments, the conductive material is in theshape of an open disk. In some embodiments, the electrode is configuredin a hollow ring shape. In some embodiments, the electrode is configuredin a hollow tube shape. In some embodiments, the array of electrodescomprises non-conductive material. In some embodiments, thenon-conductive material surrounds the conductive material within theelectrodes and serves as a physical barrier to the conductive material.In some embodiments, the conductive material within the electrodes fillsdepressions in the non-conductive material of the array. In someembodiments, the array of electrodes is configured in three-dimensions.In some embodiments, the conductive material within the electrodes isconfigured at an angle. In some embodiments, the conductive materialwithin the electrodes is configured into a hollow triangular tube. Insome embodiments, the conductive material within the electrodes isconfigured into angles between neighboring planar electrode surfaces ofless than about 180 degrees. In some embodiments, the conductivematerial configured into angles between neighboring planar electrodesurfaces of equal to or less than 180 degrees. In some embodiments, theconductive material within the electrodes is configured into angles ofmore than about or equal to 60 degrees. In some embodiments, theconductive material configured into angles between neighboring planarelectrode surfaces of equal to or more than 60 degrees. In someembodiments, the conductive material within the electrodes is configuredinto a depressed concave shape. In some embodiments, thethree-dimensional configuration of the conductive material increases thetotal surface area of the conductive material within the electrodes. Insome embodiments, the individual electrodes are about 40 μm to about 100μm in diameter. In some embodiments, the electrodes are in anon-circular configuration. In some embodiments, the angle oforientation between non-circular configurations is between about 25 and90 degrees. In some embodiments, the non-circular configurationcomprises a wavy line configuration, wherein the configuration comprisesa repeating unit comprising the shape of a pair of dots connected bylinker, wherein the linker tapers inward toward the midpoint between thepair of dots, wherein the diameters of the dots are the widest pointsalong the length of the repeating unit, wherein the edge to edgedistance between a parallel set of repeating units is equidistant, orroughly equidistant.

In some embodiments, the (AC) electrodes in the array comprise one ormore floating electrodes. The floating electrodes are not energized toestablish AC electrokinetic regions. In some embodiments, a floatingelectrode surrounds an AC electrode. In further embodiments, thefloating electrodes in the array induce an electric field with a highergradient than an electric field induced by non-floating electrodes inthe array.

In another aspect, disclosed herein, in some embodiments, is a methodfor isolating a nanoscale analyte in a sample, the method comprising: a.applying the sample to a device, the device comprising an array ofelectrodes capable of establishing an AC electrokinetic field regionwherein the electrodes comprise conductive material configured on oraround the electrodes which reduces, disrupts or alters fluid flowaround or within the vicinity of the electrodes as compared to fluidflow in regions between or substantially beyond the electrode vicinity;b. producing at least one AC electrokinetic field region, wherein the atleast one AC electrokinetic field region is a dielectrophoretic highfield region; and c. isolating the nanoscale analyte in thedielectrophoretic high field region. In some embodiments, the conductivematerial is substantially absent from the center of the individualelectrodes in the array. In some embodiments, the conductive material ispresent at the edges of the individual electrodes in the electrodearray. In some embodiments, the conductive material is in the shape ofan open disk. In some embodiments, the electrode is configured in ahollow ring shape. In some embodiments, the electrode is configured in ahollow tube shape. In some embodiments, a reduction in conductivematerial within the electrodes results in reduced fluid flow in andaround the electrode surface, leading to an increase in nanoscaleanalyte capture on the surface of the electrode. In some embodiments,the increase in nanoscale analyte capture is at least 10%, at least 20%,at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, atleast 80%, at least 90% or at least 100% or more nanoscale analytecaptured than if using conventional electrode configuration or designswithout a reduction in conductive material within the electrodes. Insome embodiments, the array of electrodes comprises non-conductivematerial. In some embodiments, the non-conductive material surrounds theconductive material within the electrodes and serves as a physicalbarrier to the conductive material. In some embodiments, the conductivematerial within the electrodes fills depressions in the non-conductivematerial of the array. In some embodiments, the array of electrodes isconfigured in three-dimensions. In some embodiments, the conductivematerial within the electrodes is configured at an angle. In someembodiments, the conductive material within the electrodes is configuredinto a hollow triangular tube. In some embodiments, the conductivematerial within the electrodes is configured into angles betweenneighboring planar electrode surfaces of less than about 180 degrees. Insome embodiments, the conductive material configured into angles betweenneighboring planar electrode surfaces of equal to or less than 180degrees. In some embodiments, the conductive material within theelectrodes is configured into angles of more than about 60 degrees. Insome embodiments, the conductive material configured into angles betweenneighboring planar electrode surfaces of equal to or more than 60degrees. In some embodiments, the conductive material within theelectrodes is configured into a depressed concave shape. In someembodiments, the three-dimensional configuration of the conductivematerial increases the total surface area of the conductive materialwithin the electrodes. In some embodiments, the individual electrodesare about 40 μm to about 100 μm in diameter. In some embodiments, theelectrodes are in a non-circular configuration. In some embodiments, theangle of orientation between non-circular configurations is betweenabout 25 and 90 degrees. In some embodiments, the non-circularconfiguration comprises a wavy line configuration, wherein theconfiguration comprises a repeating unit comprising the shape of a pairof dots connected by linker, wherein the linker tapers inward toward themidpoint between the pair of dots, wherein the diameters of the dots arethe widest points along the length of the repeating unit, wherein theedge to edge distance between a parallel set of repeating units isequidistant, or roughly equidistant. In some embodiments, the ACelectrokinetic field is produced using an alternating current having avoltage of 1 volt to 40 volts peak-peak, and/or a frequency of 5 Hz to5,000,000 Hz and duty cycles from 5% to 50%. In some embodiments, thesample comprises a fluid. In some embodiments, the conductivity of thefluid is less than or equal to 300 mS/m. In some embodiments, theconductivity of the fluid is greater than or equal to 300 mS/m. In someembodiments, the electrodes are selectively energized to provide thefirst dielectrophoretic high field region and subsequently orcontinuously selectively energized to provide the seconddielectrophoretic high field region. In some embodiments, the nanoscaleanalyte is a nucleic acid. In some embodiments, the isolated nucleicacid comprises less than about 10% non-nucleic acid cellular material orcellular protein by mass. In some embodiments, the fluid comprisescells. In some embodiments, the method further comprises lysing cells onthe array. In some embodiments, the cells are lysed using a directcurrent, a chemical lysing agent, an enzymatic lysing agent, heat,pressure, sonic energy, or a combination thereof. In some embodiments,the method further comprises degradation of residual proteins after celllysis. In some embodiments, the cells are lysed using a direct currentwith a voltage of 1-500 volts, a pulse frequency of 0.2 to 200 Hz withduty cycles from 10-50%, and a pulse duration of 0.01 to 10 secondsapplied at least once. In some embodiments, the array of electrodes isspin-coated with a hydrogel having a thickness between about 0.1 micronsand 1 micron. In some embodiments, the hydrogel is deposited onto thearray of electrodes by chemical vapor deposition or surface-initiatedpolymerization. In yet other embodiments, the hydrogel is deposited ontothe array of electrodes by dip coating, spray coating, inkjet printing,Langmuir-Blodgett coating, or combinations thereof. In still otherembodiments, the hydrogel is deposited onto the array of electrodes bygrafting of polymers by end-functionalized groups or by self-assemblyfrom solution thru solvent selectivity.

In some embodiments, the hydrogel comprises two or more layers of asynthetic polymer. In some embodiments, the hydrogel has a viscositybetween about 0.5 cP to about 5 cP prior to spin-coating or depositiononto the array of electrodes. In some embodiments, the hydrogel has aconductivity between about 0.1 S/m to about 1.0 S/m. In someembodiments, the method is completed in less than 10 minutes. In someembodiments, the array of electrodes comprises a passivation layer witha relative electrical permittivity from about 2.0 to about 4.0.

In some embodiments, the electrodes comprise one or more floatingelectrodes. The floating electrodes are not energized to establish ACelectrokinetic regions. A floating electrode surrounds an energizedelectrode. In some embodiments, the floating electrodes in the arrayinduce an electric field with a higher gradient than an electric fieldinduced by non-floating electrodes in the array.

All publications, patents, and patent applications mentioned in thisspecification are herein incorporated by reference to the same extent asif each individual publication, patent, or patent application wasspecifically and individually indicated to be incorporated by reference.

BRIEF DESCRIPTION OF THE DRAWINGS

The novel features of the invention are set forth with particularity inthe appended claims. A better understanding of the features andadvantages of the present invention will be obtained by reference to thefollowing detailed description that sets forth illustrative embodiments,in which the principles of the invention are utilized, and theaccompanying drawings of which:

FIG. 1 exemplifies a standard electrode configuration in the shape of ahollow disk. The electrode comprises conductive material around theedges of the electrode. The color filled electrodes represent the anodesand the non-color filled electrodes represent the cathodes.

FIG. 2 exemplifies an electrode configuration in the shape of a hollowring. The electrode comprises conductive material around the edges ofthe electrode. The color filled electrodes represent the anodes and thenon-color filled electrodes represent the cathodes.

FIG. 3 exemplifies an electrode configuration, wherein the electrodesare in a wavy line configuration, wherein the configuration comprises arepeating unit comprising the shape of a pair of dots connected by alinker, wherein the linker tapers inward toward the midpoint between thepair of dots, wherein the diameters of the dots are the widest pointsalong the length of the repeating unit, wherein the edge to edgedistance between a parallel set of repeating units is equidistant, orroughly equidistant. The electrode comprises conductive material onevery other wavy line configuration. The color filled electrodesrepresent the anodes and the non-color filled electrodes represent thecathodes.

FIG. 4 exemplifies an electrode configuration in the shape of acontinuous hollow wavy line configuration. The electrodes compriseconductive material around the edges of the electrode. The color filledelectrodes represent the anodes and the non-color filled electrodesrepresent the cathodes.

FIG. 5 exemplifies an array of electrodes wherein the electrodes areconfigured in the shape of a hollow ring with an extruded center. Theelectrodes comprise conductive material around the edges of theelectrodes. The exemplified ring has a 10 μm annulus of exposedplatinum. The color filled electrodes represent the anodes and thenon-color filled electrodes represent the cathodes.

FIG. 6 exemplifies a bright field image of a microlectrode arraycomprising electrodes in a hollow disk configuration in an unknownsample chamber. The disks comprised exposed platinum. The “black dots”that appear in the image are red blood cells.

FIG. 7 exemplifies a fluorescent image of the microlectrode hollow diskarray in the unknown sample chamber with nanoscale analyte isolated onthe edge of each microelectrode.

FIG. 8 exemplifies a fluorescent image of the microlectrode hollow diskarray in the unknown sample chamber with nanoscale analyte isolated onthe edge of each microelectrode at the end of the 20 minute process.

FIG. 9 exemplifies a fluorescent image of the microlectrode array in theunknown sample chamber after release of the nanoscale analyte from theedges of the electrode by termination of production of ACelectrokinetics.

FIG. 10 exemplifies the DEP gradient on a microelectrode hollow diskarray. The DEP gradient magnitude is represented by color. A positiveDEP zone is located on the edge of the electrodes while a negative DEPzone is located between the electrodes.

FIG. 11 exemplifies the ACET flow pattern in the electrode chamber. Themagnitude of the flow is depicted by color, where the strongest flow isseen a few microns above the chamber edge, while flow dead zones arelocated in the vortices center and in the electrode ring center, asindicated by the arrows. Stream lines exemplify the vortices formed bythe ACET effect. Red arrows indicate flow direction.

FIG. 12 exemplifiers a flow velocity profile (right) and a DEP gradient(right) generated by the microelectrode array with new floatingelectrode design.

DETAILED DESCRIPTION OF THE INVENTION

Described herein are methods, devices and systems suitable for isolatingor separating nanoscale analytes from complex samples. In specificembodiments, provided herein are methods, devices and systems forisolating or separating a nanoscale analyte from a sample comprisingother particulate material. In some aspects, the methods, devices andsystems may allow for rapid separation of particles and nanoscaleanalytes in a sample. In other aspects, the methods, devices and systemsmay allow for rapid isolation of nanoscale analytes from particles in asample. In various aspects, the methods, devices and systems may allowfor a rapid procedure that requires a minimal amount of material and/orresults in a highly purified nanoscale analyte isolated from complexfluids such as blood or environmental samples.

Provided in certain embodiments herein are methods, devices and systemsfor isolating or separating nanoscale analytes from a sample, themethods, devices, and systems comprising applying the fluid to a devicecomprising an array of electrodes as disclosed herein and being capableof generating AC electrokinetic forces (e.g., when the array ofelectrodes are energized). AC Electrokinetics (ACE) capture is afunctional relationship between the dielectrophoretic force (F_(DEP))and the flow force (F_(FLOW)) derived from the combination of ACelectrothermal (ACET) and AC electroosmostic (ACEO) flows. In someembodiments, the dielectrophoretic field generated is a component of ACelectrokinetic force effects. In other embodiments, the component of ACelectrokinetic force effects is AC electroosmosis or AC electrothermaleffects. In some embodiments the AC electrokinetic force, includingdielectrophoretic fields, comprises high-field regions (positive DEP,i.e. area where there is a strong concentration of electric field linesdue to a non-uniform electric field) and/or low-field regions (negativeDEP, i.e. area where there is a weak concentration of electric fieldlines due to a non-uniform electric field).

In specific instances, the nanoscale analytes (e.g., nucleic acid) areisolated (e.g., isolated or separated from particulate material) in afield region (e.g., a high field region) of a dielectrophoretic field.In some embodiments, the method, device, or system includes isolatingand concentrating nanoscale analytes in a high field DEP region. In someembodiments, the method, device, or system includes isolating andconcentrating nanoscale analytes in a low field DEP region The methodalso optionally includes devices and/or systems capable of performingone or more of the following steps: washing or otherwise removingresidual (e.g., cellular or proteinaceous) material from the nanoscaleanalyte (e.g., rinsing the array with water or buffer while thenanoscale analyte is concentrated and maintained within a high field DEPregion of the array), degrading residual proteins (e.g., degradationoccurring according to any suitable mechanism, such as with heat, aprotease, or a chemical), flushing degraded proteins from the nanoscaleanalyte, and collecting the nanoscale analyte. In some embodiments, theresult of the methods, operation of the devices, and operation of thesystems described herein is an isolated nanoscale analyte, optionally ofsuitable quantity and purity for further analysis or characterizationin, for example, enzymatic assays (e.g. PCR assays).

In some embodiments, the methods, devices and compositions disclosedherein utilize electrode configurations and designs to improveseparation and capture of the nanoscale analytes from particulatematerial. In some embodiments, the electrode arrays are configured suchthat fluid flow around or within the vicinity of the electrodes aredisrupted or altered, allowing the localization and/or retention ofnanoscale analytes around or within the electrode arrays. In otherembodiments, the improvement in nanoscale analyte capture is at least10%, at least 20%, at least 30%, at least 40%, at least 50%, at least60%, at least 70%, at least 80%, at least 90% or at least 100% or morenanoscale analyte captured than if using conventional electrodeconfiguration or designs, which do not have a reduction in conductivematerial within the electrodes.

In some embodiments, the array of electrodes as disclosed herein isspin-coated with a hydrogel having a thickness between about 0.1 micronsand 1 micron. In some embodiments, the hydrogel is deposited onto thearray of electrodes by chemical vapor deposition or surface-initiatedpolymerization. In yet other embodiments, the hydrogel is deposited ontothe array of electrodes by dip coating, spray coating, inkjet printing,Langmuir-Blodgett coating, or combinations thereof. In still otherembodiments, the hydrogel is deposited onto the array of electrodes bygrafting of polymers by end-functionalized groups or by self-assemblyfrom solution thru solvent selectivity. In some embodiments, thehydrogel comprises two or more layers of a synthetic polymer. In someembodiments, the hydrogel has a viscosity between about 0.5 cP to about5 cP prior to spin-coating or deposition onto the array of electrodes.In some embodiments, the hydrogel has a conductivity between about 0.1S/m to about 1.0 S/m.

In some embodiments, the isolated nanoscale analyte comprises less thanabout 10% non-nanoscale analyte by mass. In some embodiments, the methodis completed in less than 10 minutes.

In some embodiments, the method further comprises degrading residualproteins on the array. In some embodiments, the residual proteins aredegraded by one or more of a chemical degradant or an enzymaticdegradant. In some embodiments, the residual proteins are degraded byProteinase K.

In some embodiments, the nanoscale analyte is a nucleic acid. In otherembodiments, the nucleic acid is further amplified by polymerase chainreaction. In some embodiments, the nucleic acid comprises DNA, RNA, orany combination thereof. In some embodiments, the isolated nucleic acidcomprises less than about 80%, less than about 70%, less than about 60%,less than about 50%, less than about 40%, less than about 30%, less thanabout 20%, less than about 10%, less than about 5%, or less than about2% non-nucleic acid cellular material and/or protein by mass. In someembodiments, the isolated nucleic acid comprises greater than about 99%,greater than about 98%, greater than about 95%, greater than about 90%,greater than about 80%, greater than about 70%, greater than about 60%,greater than about 50%, greater than about 40%, greater than about 30%,greater than about 20%, or greater than about 10% nucleic acid by mass.In some embodiments, the method is completed in less than about onehour. In some embodiments, centrifugation is not used. In someembodiments, the residual proteins are degraded by one or more ofchemical degradation and enzymatic degradation. In some embodiments, theresidual proteins are degraded by Proteinase K. In some embodiments, theresidual proteins are degraded by an enzyme, the method furthercomprising inactivating the enzyme following degradation of theproteins. In some embodiments, the enzyme is inactivated by heat (e.g.,50 to 95° C. for 5-15 minutes). In some embodiments, the residualmaterial and the degraded proteins are flushed in separate or concurrentsteps. In some embodiments, the isolated nanoscale analyte is collectedby (i) turning off the second AC electrokinetic field region; and (ii)eluting the nanoscale analyte from the array in an eluant. In someembodiments, a nanoscale analyte is isolated in a form suitable forsequencing. In some embodiments, the nanoscale analyte is isolated in afragmented form suitable for shotgun-sequencing.

In some embodiments, the nucleic acid is sequenced by Sanger sequencing,pyrosequencing, ion semiconductor sequencing, polony sequencing,sequencing by ligation, DNA nanoball sequencing, sequencing by ligation,or single molecule sequencing. In some embodiments, the method furthercomprises performing a reaction on the DNA (e.g., fragmentation,restriction digestion, ligation) that is isolated and eluted from thedevices disclosed herein. In some embodiments, the reaction occurs on ornear the array or in the device. In some embodiments, the fluid orbiological sample comprises no more than 10,000 cells.

In some embodiments, the sample is a biological sample and has a lowconductivity or a high conductivity. In some embodiments, the samplecomprises a bodily fluid, blood, serum, plasma, urine, saliva, a food, abeverage, a growth medium, an environmental sample, a liquid, water,clonal cells, or a combination thereof. In some embodiments, the cellscomprise clonal cells, pathogen cells, bacteria cells, viruses, plantcells, animal cells, insect cells, and/or combinations thereof.

In some embodiments, the devices and methods disclosed herein furthercomprises using at least one of an elution tube, a chamber and areservoir to perform amplification of isolated nucleic acids as thenanoscale analyte. In some embodiments, amplification of the isolatedand eluted nucleic acid is polymerase chain reaction (PCR)-based. Insome embodiments, amplification of the nucleic acid is performed in aserpentine microchannel comprising a plurality of temperature zones. Insome embodiments, amplification is performed in aqueous dropletsentrapped in immiscible fluids (i.e., digital PCR). In some embodiments,the thermocycling comprises convection. In some embodiments, the devicecomprises a surface contacting or proximal to the electrodes, whereinthe surface is functionalized with biological ligands that are capableof selectively capturing biomolecules. In some embodiments, the surfaceselectively captures biomolecules by: a. nucleic acid hybridization; b.antibody—antigen interactions; c. biotin—avidin interactions; d. ionicor electrostatic interactions; or e. any combination thereof. In someembodiments, the surface is functionalized to minimize and/or inhibitnonspecific binding interactions by: a. polymers (e.g., polyethyleneglycol PEG); b. ionic or electrostatic interactions; c. surfactants; ord. any combination thereof. In some embodiments, the device comprises aplurality of microelectrode devices oriented (a) flat side by side, (b)facing vertically, or (c) facing horizontally. In some embodiments, thedevice comprises a module capable of performing Sanger sequencing. Insome embodiments, the module capable of performing Sanger sequencingcomprises a module capable of capillary electrophoresis, a modulecapable of multi-color fluorescence detection, or a combination thereof.

In some instances, it is advantageous that the methods described hereinare performed in a short amount of time, the devices are operated in ashort amount of time, and the systems are operated in a short amount oftime. In some embodiments, the period of time is short with reference tothe “procedure time” measured from the time between adding the fluid tothe device and obtaining isolated nanoscale analyte. In someembodiments, the procedure time is less than 3 hours, less than 2 hours,less than 1 hour, less than 30 minutes, less than 20 minutes, less than10 minutes, or less than 5 minutes.

In another aspect, the period of time is short with reference to the“hands-on time” measured as the cumulative amount of time that a personmust attend to the procedure from the time between adding the fluid tothe device and obtaining isolated nanoscale analyte. In someembodiments, the hands-on time is less than 20 minutes, less than 10minutes, less than 5 minute, less than 1 minute, or less than 30seconds.

In some instances, it is advantageous that the devices described hereincomprise a single vessel, the systems described herein comprise a devicecomprising a single vessel and the methods described herein can beperformed in a single vessel, e.g., in a dielectrophoretic device asdescribed herein. In some aspects, such a single-vessel embodimentminimizes the number of fluid handling steps and/or is performed in ashort amount of time. In some instances, the present methods, devicesand systems are contrasted with methods, devices and systems that useone or more centrifugation steps and/or medium exchanges. In someinstances, centrifugation increases the amount of hands-on time requiredto isolate nanoscale analytes. In another aspect, the single-vesselprocedure or device isolates nanoscale analytes using a minimal amountof consumable reagents.

Devices and Systems

In some embodiments, described herein are devices for isolating,purifying and collecting a nanoscale analyte from a sample. In oneaspect, described herein are devices for isolating, purifying andcollecting or eluting a nanoscale from a complex sample otherparticulate material, including cells and the like. In other aspects,the devices disclosed herein are capable of isolating, purifying,collecting and/or eluting nanoscale analytes from a sample comprisingcellular or protein material. In yet other aspects, the devicesdisclosed herein are capable of isolating, purifying, collecting and/oreluting nanoscale analytes from samples comprising a complex mixture oforganic and inorganic materials. In some aspects, the devices disclosedherein are capable of isolating, purifying, collecting and/or elutingnanoscale analytes from samples comprising organic materials. In yetother aspects, the devices disclosed herein are capable of isolating,purifying, collecting and/or eluting nanoscale analytes from samplescomprising inorganic materials.

In some embodiments, disclosed herein is a device for isolating ananoscale analyte in a sample, the device comprising: a. a housing; b. aheater and/or a reservoir comprising a protein degradation agent; and c.a plurality of alternating current (AC) electrodes as disclosed hereinwithin the housing, the AC electrodes configured to be selectivelyenergized to establish AC electrokinetic high field and ACelectrokinetic low field regions, wherein the electrodes compriseconductive material configured on or around the electrodes whichreduces, disrupts or alters fluid flow around or within the vicinity ofthe electrodes as compared to fluid flow in regions between orsubstantially beyond the electrode vicinity. In some embodiments, theconductive material is substantially absent from the center of theindividual electrodes in the array. In some embodiments, the conductivematerial is present at the edges of the individual electrodes in theelectrode array.

In some embodiments, an AC electrokinetic field is generated to collect,separate or isolate nanoscale analytes. In some embodiments, thenanoscale analytes are biomolecules, such as nucleic acids. In someembodiments, the AC electrokinetic field is a dielectrophoretic field.Accordingly, in some embodiments dielectrophoresis (DEP) is utilized invarious steps of the methods and devices described herein.

Accordingly provided herein are systems and devices comprising aplurality of alternating current (AC) electrodes as disclosed herein,the AC electrodes configured to be selectively energized to establish adielectrophoretic (DEP) field region. In some aspects, the AC electrodesmay be configured to be selectively energized to establish multipledielectrophoretic (DEP) field regions, including dielectrophoretic (DEP)high field and dielectrophoretic (DEP) low field regions. In someinstances, AC electrokinetic effects provide for concentration of largerparticulate material in low field regions and/or concentration (orcollection or isolation) of nanoscale analytes (e.g., macromolecules,such as nucleic acid) in high field regions of the DEP field. Forexample, further description of the electrodes and the concentration ofcells in DEP fields may be found in PCT patent publication WO2009/146143 A2, which is incorporated herein for such disclosure.

In specific embodiments, DEP is used to concentrate nanoscale analytesand larger particulate matter either concurrently or at different times.In certain embodiments, methods and devices described herein are capableof energizing the array of electrodes as disclosed herein so as toproduce at least one DEP field. In other embodiments, the methods anddevices described here further comprise energizing the array ofelectrodes so as to produce a first, second, and any further optionalDEP fields. In some embodiments, the devices and systems describedherein are capable of being energized so as to produce a first, second,and any further optional DEP fields.

DEP is a phenomenon in which a force is exerted on a dielectric particlewhen it is subjected to a non-uniform electric field. Depending on thestep of the methods described herein, aspects of the devices and systemsdescribed herein, and the like, the dielectric particle in variousembodiments herein is a biological nanoscale analyte, such as a nucleicacid molecule. Different steps of the methods described herein oraspects of the devices or systems described herein may be utilized toisolate and separate different components, such as intact cells or otherparticular material; further, different field regions of the DEP fieldmay be used in different steps of the methods or aspects of the devicesand systems described herein. The dielectrophoretic force generated inthe device does not require the particle to be charged. In someinstances, the strength of the force depends on the medium and thespecific particles' electrical properties, on the particles' shape andsize, as well as on the frequency of the electric field. In someinstances, fields of a particular frequency selectively manipulateparticles. In certain aspects described herein, these processes allowfor the separation of nanoscale analytes, including nucleic acidmolecules, from other components, such as cells and proteinaceousmaterial.

Also provided herein are systems and devices comprising a plurality ofdirect current (DC) electrodes. In some embodiments, the plurality of DCelectrodes comprises at least two rectangular electrodes, spreadthroughout the array. In some embodiments, the electrodes are located atthe edges of the array. In some embodiments, DC electrodes areinterspersed between AC electrodes.

In some embodiments, disclosed herein is a device for isolating ananoscale analyte in a sample, the device comprising: (1) a housing; (2)a plurality of alternating current (AC) electrodes as disclosed hereinwithin the housing, the AC electrodes configured to be selectivelyenergized to establish AC electrokinetic high field and ACelectrokinetic low field regions, whereby AC electrokinetic effectsprovide for concentration of the nanoscale analytes cells in anelectrokinetic field region of the device. In some embodiments, theplurality of electrodes is configured to be selectively energized toestablish a dielectrophoretic high field and dielectrophoretic low fieldregions.

In some embodiments, disclosed herein is a device comprising: (1) aplurality of alternating current (AC) electrodes as disclosed herein,the AC electrodes configured to be selectively energized to establish ACelectrokinetic high field and AC electrokinetic low field regions; and(2) a module capable of performing enzymatic reactions, such aspolymerase chain reaction (PCR) or other enzymatic reaction. In someembodiments, the plurality of electrodes is configured to be selectivelyenergized to establish a dielectrophoretic high field anddielectrophoretic low field regions. In some embodiments, the device iscapable of isolating a nanoscale analyte from a sample, collecting oreluting the nanoscale analyte and further performing an enzymaticreaction on the nanoscale analyte. In some embodiments, the enzymaticreaction is performed in the same chamber as the isolation and elutionstages. In other embodiments, the enzymatic reaction is performed inanother chamber than the isolation and elution stages. In still otherembodiments, a nanoscale analyte is isolated and the enzymatic reactionis performed in multiple chambers.

In some embodiments, the device further comprises at least one of anelution tube, a chamber and a reservoir to perform an enzymaticreaction. In some embodiments, the enzymatic reaction is performed in aserpentine microchannel comprising a plurality of temperature zones. Insome embodiments, the enzymatic reaction is performed in aqueousdroplets entrapped in immiscible fluids (e.g., digital PCR). In someembodiments, the thermal reaction comprises convection. In someembodiments, the device comprises a surface contacting or proximal tothe electrodes, wherein the surface is functionalized with biologicalligands that are capable of selectively capturing biomolecules.

In one aspect, described herein is a device comprising electrodes,wherein the electrodes are placed into separate chambers and DEP fieldsare created within an inner chamber by passage through pore structures.The exemplary device includes a plurality of electrodes andelectrode-containing chambers within a housing. A controller of thedevice independently controls the electrodes, as described further inPCT patent publication WO 2009/146143 A2, which is incorporated hereinfor such disclosure.

In some embodiments, chambered devices are created with a variety ofpore and/or hole structures (nanoscale, microscale and even macroscale)and contain membranes, gels or filtering materials which control,confine or prevent cells, nanoparticles or other entities from diffusingor being transported into the inner chambers while the AC/DC electricfields, solute molecules, buffer and other small molecules can passthrough the chambers.

Such devices include, but are not limited to, multiplexed electrode andchambered devices, devices that allow reconfigurable electric fieldpatterns to be created, devices that combine DC electrophoretic andfluidic processes; sample preparation devices, sample preparation,enzymatic manipulation of isolated nucleic acid molecules and diagnosticdevices that include subsequent detection and analysis, lab-on-chipdevices, point-of-care and other clinical diagnostic systems orversions.

In some embodiments, a planar electrode array device comprises a housingthrough which a sample fluid flows. In some embodiments, fluid flowsfrom an inlet end to an outlet end, optionally comprising a lateralanalyte outlet. The exemplary device includes multiple AC electrodes. Insome embodiments, the sample consists of a combination of micron-sizedentities or cells, larger nanoscale analytes and smaller nanoscaleanalytes or biomolecules.

In some embodiments, the smaller nanoscale analytes are proteins,smaller DNA, RNA and cellular fragments. In some embodiments, the planarelectrode array device is a 60×20 electrode array that is optionallysectioned into three 20×20 arrays that can be separately controlled butoperated simultaneously. The optional auxiliary DC electrodes can beswitched on to positive charge, while the optional DC electrodes areswitched on to negative charge for electrophoretic purposes. In someinstances, each of the controlled AC and DC systems is used in both acontinuous and/or pulsed manner (e.g., each can be pulsed on and off atrelatively short time intervals) in various embodiments. The optionalplanar electrode arrays along the sides of the sample flow areoptionally used to generate DC electrophoretic forces as well as AC DEP.Additionally, microelectrophoretic separation processes may beoptionally carried out, in combination with nanopore or hydrogel layerson the electrode array, using planar electrodes in the array and/orauxiliary electrodes in the x-y-z dimensions.

In various embodiments these methods, devices and systems are operatedin the AC frequency range of from 1,000 Hz to 100 MHz, at voltages whichcould range from approximately 1 volt to 2000 volts pk-pk; at DCvoltages from 1 volt to 1000 volts, at flow rates of from 10 microlitersper minute to 10 milliliter per minute, and in temperature ranges from1° C. to 120° C. In some embodiments, the methods, devices and systemsare operated in AC frequency ranges of from about 3 to about 15 kHz. Insome embodiments, the methods, devices, and systems are operated atvoltages of from 5-25 volts pk-pk. In some embodiments, the methods,devices and systems are operated at voltages of from about 1 to about 50volts/cm. In some embodiments, the methods, devices and systems areoperated at DC voltages of from about 1 to about 5 volts. In someembodiments, the methods, devices and systems are operated at a flowrate of from about 10 microliters to about 500 microliters per minute.In some embodiments, the methods, devices and systems are operated intemperature ranges of from about 20° C. to about 60° C.

In some embodiments, the methods, devices and systems are operated in ACfrequency ranges of from 1,000 Hz to 10 MHz. In some embodiments, themethods, devices and systems are operated in AC frequency ranges of from1,000 Hz to 1 MHz. In some embodiments, the methods, devices and systemsare operated in AC frequency ranges of from 1,000 Hz to 100 kHz. In someembodiments, the methods, devices and systems are operated in ACfrequency ranges of from 1,000 Hz to 10 kHz. In some embodiments, themethods, devices and systems are operated in AC frequency ranges of from10 kHz to 100 kHz. In some embodiments, the methods, devices and systemsare operated in AC frequency ranges of from 100 kHz to 1 MHz.

In some embodiments, the methods, devices and systems are operated atvoltages from approximately 1 volt to 1500 volts pk-pk. In someembodiments, the methods, devices and systems are operated at voltagesfrom approximately 1 volt to 1500 volts pk-pk. In some embodiments, themethods, devices and systems are operated at voltages from approximately1 volt to 1000 volts pk-pk. In some embodiments, the methods, devicesand systems are operated at voltages from approximately 1 volt to 500volts pk-pk. In some embodiments, the methods, devices and systems areoperated at voltages from approximately 1 volt to 250 volts pk-pk. Insome embodiments, the methods, devices and systems are operated atvoltages from approximately 1 volt to 100 volts pk-pk. In someembodiments, the methods, devices and systems are operated at voltagesfrom approximately 1 volt to 50 volts pk-pk.

In some embodiments, the methods, devices and systems are operated at DCvoltages from 1 volt to 1000 volts. In some embodiments, the methods,devices and systems are operated at DC voltages from 1 volt to 500volts. In some embodiments, the methods, devices and systems areoperated at DC voltages from 1 volt to 250 volts. In some embodiments,the methods, devices and systems are operated at DC voltages from 1 voltto 100 volts. In some embodiments, the methods, devices and systems areoperated at DC voltages from 1 volt to 50 volts.

In some embodiments, the AC electrokinetic field is produced using analternating current having a voltage of 1 volt to 40 volts peak-peak,and/or a frequency of 5 Hz to 5,000,000 Hz and duty cycles from 5% to50%.

In some embodiments, the methods, devices, and systems are operated atflow rates of from 10 microliters per minute to 1 ml per minute. In someembodiments, the methods, devices, and systems are operated at flowrates of from 10 microliters per minute to 500 microliters per minute.In some embodiments, the methods, devices, and systems are operated atflow rates of from 10 microliters per minute to 250 microliters perminute. In some embodiments, the methods, devices, and systems areoperated at flow rates of from 10 microliters per minute to 100microliters per minute.

In some embodiments, the methods, devices, and systems are operated intemperature ranges from 1° C. to 100° C. In some embodiments, themethods, devices, and systems are operated in temperature ranges from20° C. to 95° C. In some embodiments, the methods, devices, and systemsare operated in temperature ranges from 25° C. to 100° C. In someembodiments, the methods, devices, and systems are operated at roomtemperature.

In some embodiments, the controller independently controls each of theelectrodes. In some embodiments, the controller is externally connectedto the device such as by a socket and plug connection, or is integratedwith the device housing.

In some embodiments, the device comprises a housing and a heater orthermal source and/or a reservoir comprising a protein degradationagent. In some embodiments, the heater or thermal source is capable ofincreasing the temperature of the fluid to a desired temperature (e.g.,to a temperature suitable for degrading proteins, about 30° C., 40° C.,50° C., 60° C., 70° C., or the like). In some embodiments, the heater orthermal source is suitable for operation as a PCR thermocycler. In otherembodiments, the heater or thermal source is used to maintain a constanttemperature (isothermal conditions). In some embodiments, the proteindegradation agent is a protease. In other embodiments, the proteindegradation agent is Proteinase K and the heater or thermal source isused to inactivate the protein degradation agent.

In some embodiments, the device comprises a second reservoir comprisingan eluant. The eluant is any fluid suitable for eluting the isolatednanoscale analyte from the device. In some instances the eluant is wateror a buffer. In some instances, the eluant comprises reagents requiredfor a DNA sequencing method.

In some embodiments, a system or device described herein is capable ofmaintaining a constant temperature. In some embodiments, a system ordevice described herein is capable of cooling the array or chamber. Insome embodiments, a system or device described herein is capable ofheating the array or chamber. In some embodiments, a system or devicedescribed herein comprises a thermocycler. In some embodiments, thedevices disclosed herein comprise a localized temperature controlelement. In some embodiments, the devices disclosed herein are capableof both sensing and controlling temperature.

In some embodiments, the devices further comprise heating or thermalelements. In some embodiments, a heating or thermal element is localizedunderneath an electrode. In some embodiments, the heating or thermalelements comprise a metal. In some embodiments, the heating or thermalelements comprise tantalum, aluminum, tungsten, or a combinationthereof. Generally, the temperature achieved by a heating or thermalelement is proportional to the current running through it. In someembodiments, the devices disclosed herein comprise localized coolingelements. In some embodiments, heat resistant elements are placeddirectly under the exposed electrode array. In some embodiments, thedevices disclosed herein are capable of achieving and maintaining atemperature between about 20° C. and about 120° C. In some embodiments,the devices disclosed herein are capable of achieving and maintaining atemperature between about 30° C. and about 100° C. In other embodiments,the devices disclosed herein are capable of achieving and maintaining atemperature between about 20° C. and about 95° C. In some embodiments,the devices disclosed herein are capable of achieving and maintaining atemperature between about 25° C. and about 90° C., between about 25° C.and about 85° C., between about 25° C. and about 75° C., between about25° C. and about 65° C. or between about 25° C. and about 55° C. In someembodiments, the devices disclosed herein are capable of achieving andmaintaining a temperature of about 20° C., about 30° C., about 40° C.,about 50° C., about 60° C., about 70° C., about 80° C., about 90° C.,about 100° C., about 110° C. or about 120° C.

Electrodes

In some embodiments, the methods, devices and compositions disclosedherein utilize electrode configurations and designs to improveseparation and capture of the nanoscale analytes from particulatematerial. In some embodiments, the electrode arrays are configured suchthat fluid flow around or within the vicinity of the electrodes aredisrupted or altered, allowing the localization and/or retention ofnanoscale analytes around or within the electrode arrays. In otherembodiments, the improvement in nanoscale analyte capture is at least10%, at least 20%, at least 30%, at least 40%, at least 50%, at least60%, at least 70%, at least 80%, at least 90% or at least 100% or morenanoscale analyte captured than if using conventional electrodeconfiguration or designs.

In some embodiments, the conductive material is in the shape of an opendisk. In some embodiments, the electrode is configured in a hollow ringshape. In some embodiments, the electrode is configured in a hollow tubeshape. In some embodiments, the array of electrodes as disclosed hereincomprises non-conductive material. In some embodiments, thenon-conductive material surrounds the conductive material within theelectrodes and serves as a physical barrier to the conductive material.In some embodiments, the conductive material within the electrodes fillsdepressions in the non-conductive material of the array. In someembodiments, the array of electrodes as disclosed herein is configuredin three-dimensions.

In one embodiment, the array of electrodes as disclosed herein comprisesconductive material in only a fraction of the electrode array. In someembodiments, the conductive material is only present in less than about10% of the electrode array. In some embodiments, the conductive materialis only present in about 10% of the electrode array. In otherembodiments, the conductive material is only present in about 20% of theelectrode array. In still other embodiments, the conductive material isonly present in about 30% of the electrode array. In yet otherembodiments, the conductive material is only present in about 40% of theelectrode array. In still other embodiments, the conductive material isonly present in about 50% of the electrode array. In some embodiments,the conductive material is only present in about 60% of the electrodearray. In one embodiment, the conductive material is only present inabout 70% of the electrode array. In still other embodiments, theconductive material is only present in about 80% of the electrode array.In yet other embodiments, the conductive material is only present inabout 90% of the electrode array.

In still other embodiments, the conductive material is only present inabout 10%, in about 15%, in about 20%, in about 25%, in about 30%, inabout 35%, in about 40%, in about 45%, in about 50%, in about 55%, inabout 60%, in about 65%, in about 70%, in about 75%, in about 80%, inabout 85% and in about 90% of the electrode array. In yet otherembodiments, the conductive material is present in about 10-70% of theelectrode array, in about 10-60% of the electrode array, in about 10-50%of the electrode array, in about 10-40% of the electrode or in about10-30% of the electrode array. In other embodiments, the conductivematerial is present in about 30-90% of the electrode array, in about30-80% of the electrode array, in about 30-70% of the electrode array,in about 30-60% of the electrode array or in about 30-50% of theelectrode array. In some embodiments, the conductive material is presentin about 8 to about 40% of the electrode array.

In yet other embodiments, the conductive material is substantiallyabsent from the center of the individual electrodes in the electrodearray. In other embodiments, the conductive material is only present atthe edges of the individual electrodes in the electrode array. In stillother embodiments, the conductive material is in the shape of an opendisk, which comprises conductive material that is discontinuous in theopen disk electrode. In some embodiments, the electrode is a hollow ringelectrode shape, which comprises conductive material in the electrodearray that is substantially absent from the center of the individualelectrodes or is only at the edge of the individual electrodes. Thehollow ring electrode shape, like the open disk shape, reduces thesurface area of the conductive material in an electrode. The reductionin conductive material present on the electrode results in flow in andaround the electrode surface, leading to increases in nanoscale analytecaptured on the surface of the electrode.

In some embodiments, a layer of non-conductive material is present incertain areas of the electrode or in the proximal vicinity of theelectrode array. In one embodiment, a layer of non-conductive materialsurrounds the electrode array, creating a physical barrier or wallsurrounding the array. In some embodiments, the electrode array isdepressed into the array material, creating a well or depression on thearray surface wherein electrode material or substantially electrodematerial is present in the well or depression.

In some embodiments, the electrode configuration is in three-dimensions.In some embodiments, the electrode material is folded into an angleconfiguration. In other embodiments, the electrode material is formedinto a triangular tube. In other embodiments, the electrode material isformed into a hollow triangular tube. In still other embodiments, thethree dimensional electrode comprises angles between neighboring planarelectrode surfaces of less than about 180 degrees, less than about 170degrees, less than about 160 degrees, less than about 150 degrees, lessthan about 140 degrees, less than about 130 degrees, less than about 120degrees, less than about 110 degrees, less than about 100 degrees, lessthan about 90 degrees, less than about 80 degrees, less than about 70degrees, but not less than about 60 degrees. In some embodiments, theconductive material configured into angles between neighboring planarelectrode surfaces of equal to or less than 180 degrees. In someembodiments, the three dimensional electrode configuration comprisesangles between neighboring planar electrode surfaces of more than about60 degrees, more than about 70 degrees, more than about 80 degrees, morethan about 90 degrees, more than about 100 degrees, more than about 110degrees, more than about 120 degrees, more than about 130 degrees, morethan about 140 degrees, more than about 150 degrees, more than about 160degrees, more than about 170 degrees, but not more than about 180degrees. In some embodiments, the conductive material configured intoangles between neighboring planar electrode surfaces of equal to or morethan 60 degrees. In some embodiments, the conductive material within theelectrodes is configured into a depressed concave shape. In yet otherembodiments, the electrode configuration is a depressed basketelectrode. The three-dimensional structure of the electrode increasesthe total surface area of the electrode, allowing interrogation of morefluid in a defined unit of time.

In some embodiments, the individual electrodes are about 40 μm to about100 μm in diameter. In still other embodiments, the individualelectrodes are about 40 μm, about 45 μm, about 50 μm, about 55 μm, about60 μm, about 65 μm, about 70 μm, about 75 μm, about 80 μm, about 85 μm,about 90 μm, about 95 μm or about 100 μm in diameter. In yet otherembodiments, the individual electrodes are about 40 μm to about 50 μm,about 40 μm to about 60 μm or about 40 μm to about 70 μm. In still otherembodiments, the individual electrodes are about 100 μm, about 200 μm,about 300 μm, about 400 μm, about 500 μm, about 600 μm, about 700 μm,about 800 μm, about 900 μm, or about 1000 μm in diameter.

The plurality of alternating current electrodes are optionallyconfigured in any manner suitable for the separation processes describedherein. In other embodiments, the array of electrodes as disclosedherein comprises a pattern of electrode configurations, wherein theconfiguration comprises a repeating unit of electrode arrays. In someembodiments, the edge to edge distance between a parallel set ofrepeating units is equidistant, or roughly equidistant. Furtherdescription of the system or device including electrodes and/orconcentration of cells in DEP fields is found in PCT patent publicationWO 2009/146143, which is incorporated herein for such disclosure.

In some embodiments, the electrodes disclosed herein comprise anysuitable metal. In other embodiments, the electrodes disclosed hereincomprise a noble metal. In some embodiments, the electrodes can includebut are not limited to: aluminum, copper, carbon, iron, silver, gold,palladium, platinum, iridium, platinum iridium alloy, ruthenium,rhodium, osmium, tantalum, titanium, tungsten, polysilicon, and indiumtin oxide, or combinations thereof, as well as silicide materials suchas platinum silicide, titanium silicide, gold silicide, or tungstensilicide. In some embodiments, the electrodes can comprise a conductiveink capable of being screen-printed. In some embodiments, the electrodescomprise a conductive polymer, such as polyacetylene or polythiophene.

In one embodiment, the electrode material is about 100 to about 1000 nmthick. In some embodiments, the electrode material is about 200 to about800 nm thick. In yet other embodiments, the electrode material is about300 to about 500 nm thick. In still other embodiments, the electrodematerial is about 100 nm, about 150 nm, about 200 nm, about 250 nm,about 300 nm, about 350 nm, about 400 nm, about 450 nm, about 500 nm,about 550 nm, about 600 nm, about 650 nm, about 700 nm, about 750 nm,about 800 nm, about 850 nm, about 900 nm, about 950 nm or about 1000 nmthick.

In some embodiments, an adhesion layer is deposited or printed onto thearray as a protective layer prior to deposition of the electrodematerial. In some embodiments, the adhesion layer comprises any suitablematerial. In one embodiment, the adhesion layer comprises titanium ortungsten. In other embodiments, the adhesion layer is between about 10to about 50 nm thick. In some embodiments, the adhesion layer is betweenabout 20 to about 40 nm thick. In yet other embodiments, the adhesionlayer is between about 20 to about 30 nm thick. In still otherembodiments, the adhesion layer is about 10 nm, about 20 nm, about 30nm, about 40 nm or about 50 nm thick.

In some embodiments, the edge to edge (E2E) to diameter ratio of anindividual electrode is about 10 μm to about 500 μm. In someembodiments, the E2E of an electrode is about 50 μm to about 300 μm. Inyet other embodiments, the E2E of an electrode is about 100 μm to about200 μm. In still other embodiments, the E2E of an electrode is about 50μm, about 60 μm, about 70 μm, about 80 μm, about 90 μm, about 100 μm,about 110 μm, about 120 μmm about 130 μm, about 140 μm, about 150 μm,about 160 μm, about 170 μm, about 180 μm, about 190 μm, about 200 μm,about 210 μm, about 220 μm, about 230 μm, about 240 μm, about 250 μm,about 260 μm, about 270 μm, about 280 μm, about 290 μm, about 300 μm,about 310 μm, about 320 μm, about 330 μm, about 340 μm, about 350 μm,about 360 μm, about 370 μm, about 380 μm, about 390 μm, about 400 μm,about 410 μm, about 420 μm, about 430 μm, about 440 μm, about 450 μm,about 460 μm, about 470 μm, about 480 μm, about 490 μm or about 500 μm.In some embodiments, the E2E of an electrode is about 750 μm, about 1000μm, about 1500 μm, or about 2000 μm.

In some embodiments, the electrodes disclosed herein are dry-etched. Insome embodiments, the electrodes are wet etched. In some embodiments,the electrodes undergo a combination of dry etching and wet etching.

In some embodiments, each electrode is individually site-controlled.

In some embodiments, an array of electrodes as disclosed herein iscontrolled as a unit.

The array can be of any suitable material. In some embodiments, thearray comprises plastic or silica. In some embodiments, the arraycomprises silicon dioxide. In some embodiments, the array comprisesaluminum.

In some embodiments, a passivation layer is employed. In someembodiments, a passivation layer can be formed from any suitablematerial known in the art. In some embodiments, the passivation layercomprises silicon nitride. In some embodiments, the passivation layercomprises silicon dioxide. In some embodiments, the passivation layerhas a relative electrical permittivity of from about 2.0 to about 8.0.In some embodiments, the passivation layer has a relative electricalpermittivity of from about 3.0 to about 8.0, about 4.0 to about 8.0 orabout 5.0 to about 8.0. In some embodiments, the passivation layer has arelative electrical permittivity of about 2.0 to about 4.0. In someembodiments, the passivation layer has a relative electricalpermittivity of from about 2.0 to about 3.0. In some embodiments, thepassivation layer has a relative electrical permittivity of about 2.0,about 2.5, about 3.0, about 3.5 or about 4.0.

In some embodiments, the passivation layer is between about 0.1 micronsand about 10 microns in thickness. In some embodiments, the passivationlayer is between about 0.5 microns and 8 microns in thickness. In someembodiments, the passivation layer is between about 1.0 micron and 5microns in thickness. In some embodiments, the passivation layer isbetween about 1.0 micron and 4 microns in thickness. In someembodiments, the passivation layer is between about 1.0 micron and 3microns in thickness. In some embodiments, the passivation layer isbetween about 0.25 microns and 2 microns in thickness. In someembodiments, the passivation layer is between about 0.25 microns and 1micron in thickness.

In some embodiments, the passivation layer is comprised of any suitableinsulative low k dielectric material, including but not limited tosilicon nitride, silicon dioxide or titanium dioxide. In someembodiments, the passivation layer is chosen from the group consistingof polyamids, carbon, doped silicon nitride, carbon doped silicondioxide, fluorine doped silicon nitride, fluorine doped silicon dioxide,porous silicon dioxide, or any combinations thereof. In someembodiments, the passivation layer can comprise a dielectric ink capableof being screen-printed.

Electrode Geometry

In some embodiments, the electrodes disclosed herein can be arranged inany manner suitable for practicing the methods disclosed herein.

In various embodiments, a variety of configurations for the devices arepossible. For example, a device comprising a larger array of electrodes,for example in a square or rectangular pattern configured to create arepeating non-uniform electric field to enable AC electrokinetics. Forillustrative purposes only, a suitable electrode array may include, butis not limited to, a 10×10 electrode configuration, a 50×50 electrodeconfiguration, a 10×100 electrode configuration, 20×100 electrodeconfiguration, or a 20×80 electrode configuration.

In some embodiments, the electrodes are in a dot configuration, e.g. theelectrodes comprise a generally circular or round configuration (see,e.g., FIGS. 1 & 2). In some embodiments, the electrodes are configuredas disks. In some embodiments, the electrodes are configured as rings.In some embodiments, the angle of orientation between dots is from about30° to about 90° degrees. In some embodiments, the angle of orientationbetween dots is from about 25° to about 60°. In some embodiments, theangle of orientation between dots is from about 30° to about 55°. Insome embodiments, the angle of orientation between dots is from about30° to about 50°. In some embodiments, the angle of orientation betweendots is from about 35° to about 45°. In some embodiments, the angle oforientation between dots is about 25°. In some embodiments, the angle oforientation between dots is about 30°. In some embodiments, the angle oforientation between dots is about 35°. In some embodiments, the angle oforientation between dots is about 40°. In some embodiments, the angle oforientation between dots is about 45°. In some embodiments, the angle oforientation between dots is about 50°. In some embodiments, the angle oforientation between dots is about 55°. In some embodiments, the angle oforientation between dots is about 60°. In some embodiments, the angle oforientation between dots is about 65°. In some embodiments, the angle oforientation between dots is about 70°. In some embodiments, the angle oforientation between dots is about 75°. In some embodiments, the angle oforientation between dots is about 80°. In some embodiments, the angle oforientation between dots is about 85°. In some embodiments, the angle oforientation between dots is about 90°.

In other embodiments, the electrodes are in a non-circular configuration(see, e.g., FIGS. 3 & 4). In some embodiments, the angle of orientationbetween non-circular configurations is between about 25 and 90 degrees.In some embodiments, the angle of orientation between non-circularconfigurations is from about 30° to about 90° degrees. In someembodiments, the angle of orientation between non-circularconfigurations is from about 25° to about 60°. In some embodiments, theangle of orientation between non-circular configurations is from about30° to about 55°. In some embodiments, the angle of orientation betweennon-circular configurations is from about 30° to about 50°. In someembodiments, the angle of orientation between non-circularconfigurations is from about 35° to about 45°. In some embodiments, theangle of orientation between non-circular configurations is about 25°.In some embodiments, the angle of orientation between non-circularconfigurations is about 30°. In some embodiments, the angle oforientation between non-circular configurations is about 35°. In someembodiments, the angle of orientation between non-circularconfigurations is about 40°. In some embodiments, the angle oforientation between non-circular configurations is about 45°. In someembodiments, the angle of orientation between non-circularconfigurations is about 50°. In some embodiments, the angle oforientation between non-circular configurations is about 55°. In someembodiments, the angle of orientation between non-circularconfigurations is about 60°. In some embodiments, the angle oforientation between non-circular configurations is about 65°. In someembodiments, the angle of orientation between non-circularconfigurations is about 70°. In some embodiments, the angle oforientation between non-circular configurations is about 75°. In someembodiments, the angle of orientation between non-circularconfigurations is about 80°. In some embodiments, the angle oforientation between non-circular configurations is about 85°. In someembodiments, the angle of orientation between non-circularconfigurations is about 90°.

In some embodiments, the electrodes are in a substantially elongatedconfiguration.

In some embodiments, the electrodes are in a configuration resemblingwavy or nonlinear lines (see, e.g., FIGS. 3 & 4). In some embodiments,the array of electrodes is in a wavy or nonlinear line configuration,wherein the configuration comprises a repeating unit comprising theshape of a pair of dots connected by a linker, wherein the dots andlinker define the boundaries of the electrode, wherein the linker tapersinward towards or at the midpoint between the pair of dots, wherein thediameters of the dots are the widest points along the length of therepeating unit, wherein the edge to edge distance between a parallel setof repeating units is equidistant, or roughly equidistant. In someembodiments, the electrodes are strips resembling wavy lines. In someembodiments, the edge to edge distance between the electrodes isequidistant, or roughly equidistant throughout the wavy lineconfiguration. In some embodiments, the use of wavy line electrodes, asdisclosed herein, lead to an enhanced DEP field gradient.

In some embodiments, the electrodes disclosed herein are in a planarconfiguration. In some embodiments, the electrodes disclosed herein arein a non-planar configuration (see, e.g., FIG. 5).

In some embodiments, the devices disclosed herein surface selectivelycaptures nanoscale biomolecules on its surface. For example, the devicesdisclosed herein may capture nanoscale analytes such as nucleic acids,by, for example, a. nucleic acid hybridization; b. antibody—antigeninteractions; c. biotin—avidin interactions; d. ionic or electrostaticinteractions; or e. any combination thereof. The devices disclosedherein, therefore, may incorporate a functionalized surface whichincludes capture molecules, such as complementary nucleic acid probes,antibodies or other protein captures capable of capturing biomolecules(such as nucleic acids), biotin or other anchoring captures capable ofcapturing complementary target molecules such as avidin, capturemolecules capable of capturing biomolecules (such as nucleic acids) byionic or electrostatic interactions, or any combination thereof.

In some embodiments, the surface is functionalized to minimize and/orinhibit nonspecific binding interactions by: a. polymers (e.g.,polyethylene glycol PEG); b. ionic or electrostatic interactions; c.surfactants; or d. any combination thereof. In some embodiments, themethods disclosed herein include use of additives which reducenon-specific binding interactions by interfering in such interactions,such as Tween 20 and the like, bovine serum albumin, nonspecificimmunoglobulins, etc.

In some embodiments, the device comprises a plurality of microelectrodedevices oriented (a) flat side by side, (b) facing vertically, or (c)facing horizontally. In other embodiments, the electrodes are in asandwiched configuration, e.g. stacked on top of each other in avertical format.

Hydrogels

Overlaying electrode structures with one or more layers of materials canreduce the deleterious electrochemistry effects, including but notlimited to electrolysis reactions, heating, and chaotic fluid movementthat may occur on or near the electrodes, and still allow the effectiveseparation of cells, bacteria, virus, nanoparticles, DNA, and otherbiomolecules to be carried out. In some embodiments, the materialslayered over the electrode structures may be one or more porous layers.In other embodiments, the one or more porous layers is a polymer layer.In other embodiments, the one or more porous layers is a hydrogel.

In general, the hydrogel should have sufficient mechanical strength andbe relatively chemically inert such that it will be able to endure theelectrochemical effects at the electrode surface withoutdisconfiguration or decomposition. In general, the hydrogel issufficiently permeable to small aqueous ions, but keeps biomoleculesaway from the electrode surface.

In some embodiments, the hydrogel is a single layer, or coating.

In some embodiments, the hydrogel comprises a gradient of porosity,wherein the bottom of the hydrogel layer has greater porosity than thetop of the hydrogel layer.

In some embodiments, the hydrogel comprises multiple layers or coatings.In some embodiments, the hydrogel comprises two coats. In someembodiments, the hydrogel comprises three coats. In some embodiments,the bottom (first) coating has greater porosity than subsequentcoatings. In some embodiments, the top coat is has less porosity thanthe first coating. In some embodiments, the top coat has a mean porediameter that functions as a size cut-off for particles of greater than100 picometers in diameter.

In some embodiments, the hydrogel has a conductivity from about 0.001S/m to about 10 S/m. In some embodiments, the hydrogel has aconductivity from about 0.01 S/m to about 10 S/m. In some embodiments,the hydrogel has a conductivity from about 0.1 S/m to about 10 S/m. Insome embodiments, the hydrogel has a conductivity from about 1.0 S/m toabout 10 S/m. In some embodiments, the hydrogel has a conductivity fromabout 0.01 S/m to about 5 S/m. In some embodiments, the hydrogel has aconductivity from about 0.01 S/m to about 4 S/m. In some embodiments,the hydrogel has a conductivity from about 0.01 S/m to about 3 S/m. Insome embodiments, the hydrogel has a conductivity from about 0.01 S/m toabout 2 S/m. In some embodiments, the hydrogel has a conductivity fromabout 0.1 S/m to about 5 S/m. In some embodiments, the hydrogel has aconductivity from about 0.1 S/m to about 4 S/m. In some embodiments, thehydrogel has a conductivity from about 0.1 S/m to about 3 S/m. In someembodiments, the hydrogel has a conductivity from about 0.1 S/m to about2 S/m. In some embodiments, the hydrogel has a conductivity from about0.1 S/m to about 1.5 S/m. In some embodiments, the hydrogel has aconductivity from about 0.1 S/m to about 1.0 S/m.

In some embodiments, the hydrogel has a conductivity of about 0.1 S/m.In some embodiments, the hydrogel has a conductivity of about 0.2 S/m.In some embodiments, the hydrogel has a conductivity of about 0.3 S/m.In some embodiments, the hydrogel has a conductivity of about 0.4 S/m.In some embodiments, the hydrogel has a conductivity of about 0.5 S/m.In some embodiments, the hydrogel has a conductivity of about 0.6 S/m.In some embodiments, the hydrogel has a conductivity of about 0.7 S/m.In some embodiments, the hydrogel has a conductivity of about 0.8 S/m.In some embodiments, the hydrogel has a conductivity of about 0.9 S/m.In some embodiments, the hydrogel has a conductivity of about 1.0 S/m.

In some embodiments, the hydrogel has a thickness from about 0.1 micronsto about 10 microns. In some embodiments, the hydrogel has a thicknessfrom about 0.1 microns to about 5 microns. In some embodiments, thehydrogel has a thickness from about 0.1 microns to about 4 microns. Insome embodiments, the hydrogel has a thickness from about 0.1 microns toabout 3 microns. In some embodiments, the hydrogel has a thickness fromabout 0.1 microns to about 2 microns. In some embodiments, the hydrogelhas a thickness from about 1 micron to about 5 microns. In someembodiments, the hydrogel has a thickness from about 1 micron to about 4microns. In some embodiments, the hydrogel has a thickness from about 1micron to about 3 microns. In some embodiments, the hydrogel has athickness from about 1 micron to about 2 microns. In some embodiments,the hydrogel has a thickness from about 0.5 microns to about 1 micron.

In some embodiments, the viscosity of a hydrogel solution prior tospin-coating or deposition onto the array of electrodes ranges fromabout 0.5 cP to about 5 cP. In some embodiments, a single coating ofhydrogel solution has a viscosity of between about 0.75 cP and 5 cPprior to spin-coating or deposition onto the array of electrodes. Insome embodiments, in a multi-coat hydrogel, the first hydrogel solutionhas a viscosity from about 0.5 cP to about 1.5 cP prior to spin coatingor deposition onto the array of electrodes. In some embodiments, thesecond hydrogel solution has a viscosity from about 1 cP to about 3 cP.The viscosity of the hydrogel solution is based on the polymersconcentration (0.1%-10%) and polymers molecular weight (10,000 to300,000) in the solvent and the starting viscosity of the solvent.

In some embodiments, the first hydrogel coating has a thickness betweenabout 0.5 microns and 1 micron. In some embodiments, the first hydrogelcoating has a thickness between about 0.5 microns and 0.75 microns. Insome embodiments, the first hydrogel coating has a thickness betweenabout 0.75 and 1 micron. In some embodiments, the second hydrogelcoating has a thickness between about 0.2 microns and 0.5 microns. Insome embodiments, the second hydrogel coating has a thickness betweenabout 0.2 and 0.4 microns. In some embodiments, the second hydrogelcoating has a thickness between about 0.2 and 0.3 microns. In someembodiments, the second hydrogel coating has a thickness between about0.3 and 0.4 microns.

In some embodiments, the hydrogel comprises any suitable syntheticpolymer forming a hydrogel. In general, any sufficiently hydrophilic andpolymerizable molecule may be utilized in the production of a syntheticpolymer hydrogel for use as disclosed herein. Polymerizable moieties inthe monomers may include alkenyl moieties including but not limited tosubstituted or unsubstituted α,β, unsaturated carbonyls wherein thedouble bond is directly attached to a carbon which is double bonded toan oxygen and single bonded to another oxygen, nitrogen, sulfur,halogen, or carbon; vinyl, wherein the double bond is singly bonded toan oxygen, nitrogen, halogen, phosphorus or sulfur; allyl, wherein thedouble bond is singly bonded to a carbon which is bonded to an oxygen,nitrogen, halogen, phosphorus or sulfur; homoallyl, wherein the doublebond is singly bonded to a carbon which is singly bonded to anothercarbon which is then singly bonded to an oxygen, nitrogen, halogen,phosphorus or sulfur; alkynyl moieties wherein a triple bond existsbetween two carbon atoms. In some embodiments, acryloyl or acrylamidomonomers such as acrylates, methacrylates, acrylamides, methacrylamides,etc., are useful for formation of hydrogels as disclosed herein. Morepreferred acrylamido monomers include acrylamides, N-substitutedacrylamides, N-substituted methacrylamides, and methacrylamide. In someembodiments, a hydrogel comprises polymers such as epoxide-basedpolymers, vinyl-based polymers, allyl-based polymers, homoallyl-basedpolymers, cyclic anhydride-based polymers, ester-based polymers,ether-based polymers, alkylene-glycol based polymers (e.g.,polypropylene glycol), and the like.

In some embodiments, the hydrogel comprises poly(2-hydroxyethylmethacrylate) (pHEMA), cellulose acetate, celluloseacetate phthalate, cellulose acetate butyrate, or any appropriateacrylamide or vinyl-based polymer, or a derivative thereof.

In some embodiments, the hydrogel is applied by vapor deposition.

In some embodiments, the hydrogel is polymerized via atom-transferradical-polymerization (ATRP).

In some embodiments, the hydrogel is polymerized via ActivatorsReGenerated by Electron Transfer-polymerization (ARGET).

In some embodiments, the hydrogel is polymerized via Initiators forContinuous Activator Regeneration-polymerization (ICAR).

In some embodiments, the hydrogel is polymerized via Nitroxide-MediatedRadical Polymerization (NMP)

In some embodiments, the hydrogel is polymerized viaPhotoinitiated-ATRP.

In some embodiments, the hydrogel is polymerized via reversibleaddition-fragmentation chain-transfer (RAFT) polymerization.

In some embodiments, additives are added to a hydrogel to increaseconductivity of the gel. In some embodiments, hydrogel additives areconductive polymers (e.g., PEDOT: PSS), salts (e.g., copper chloride),metals (e.g., gold), plasticizers (e.g., PEG200, PEG 400, or PEG 600),or co-solvents.

In some embodiments, the hydrogel also comprises compounds or materialswhich help maintain the stability of the DNA hybrids, including, but notlimited to histidine, histidine peptides, polyhistidine, lysine, lysinepeptides, and other cationic compounds or substances.

In various embodiments provided herein, a method described hereincomprises producing a DEP field region and optionally a second DEP fieldregion with the array. In various embodiments provided herein, a deviceor system described herein is capable of producing a DEP field regionand optionally a second DEP field region with the array. In someinstances, the first and second field regions are part of a single field(e.g., the first and second regions are present at the same time, butare found at different locations within the device and/or upon thearray). In some embodiments, the first and second field regions aredifferent fields (e.g. the first region is created by energizing theelectrodes at a first time, and the second region is created byenergizing the electrodes a second time). In specific aspects, the DEPfield region is suitable for concentrating or isolating cells (e.g.,into a low field DEP region). In some embodiments, the optional secondDEP field region is suitable for concentrating smaller particles, suchas molecules (e.g., nucleic acid), for example into a high field DEPregion. In some instances, a method described herein optionally excludesuse of either the first or second DEP field region.

In some embodiments, the DEP field region is in the same chamber of adevice as disclosed herein as the optional second DEP field region. Insome embodiments, the DEP field region and the optional second DEP fieldregion occupy the same area of the array of electrodes.

In some embodiments, the DEP field region is in a separate chamber of adevice as disclosed herein, or a separate device entirely, from thesecond DEP field region.

DEP Field Region

In some aspects, e.g., high conductance buffers (>100 mS/m), the methoddescribed herein comprises applying a sample comprising nanoscaleanalytes and other particulate material to a device comprising an arrayof electrodes as disclosed herein, and, thereby, isolating andcollecting the nanoscale analytes in a DEP field region. In someaspects, the devices and systems described herein are capable ofapplying a sample comprising nanoscale analytes and other particulatematerial to the device comprising an array of electrodes as disclosedherein, and, thereby, isolating and collecting the nanoscale analytes ina DEP field region. Subsequent or concurrent second, or optional thirdand fourth DEP regions, may collect or isolate other sample components,including intact cells and other particulate material.

The DEP field region generated may be any field region suitable forisolating and collecting nanoscale analytes from a sample. For thisapplication, the nanoscale analytes are generally concentrated near thearray of electrodes as disclosed herein. In some embodiments, the DEPfield region is a dielectrophoretic low field region. In someembodiments, the DEP field region is a dielectrophoretic high fieldregion. In some aspects, e.g. low conductance buffers (<100 mS/m), themethod described herein comprises applying a fluid comprising cells to adevice comprising an array of electrodes as disclosed herein, and,thereby, concentrating the nanoscale analytes in a DEP field region.

In some aspects, the devices and systems described herein are capable ofapplying a sample comprising nanoscale analytes and other particulatematerial to the device comprising an array of electrodes as disclosedherein, and concentrating the nanoscale analytes in a DEP field region.In some embodiments, the nanoscale analytes are captured in adielectrophoretic high field region. In some embodiments, the nanoscaleanalytes are captured in a dielectrophoretic low-field region. Highversus low field capture is generally dependent on the conductivity ofthe fluid, wherein generally, the crossover point between high and lowconductivity fluid is between about 300-500 mS/m. In some embodiments,the DEP field region is a dielectrophoretic low field region performedin fluid conductivity of greater than about 300 mS/m. In someembodiments, the DEP field region is a dielectrophoretic low fieldregion performed in fluid conductivity of less than about 300 mS/m. Insome embodiments, the DEP field region is a dielectrophoretic high fieldregion performed in fluid conductivity of greater than about 300 mS/m.In some embodiments, the DEP field region is a dielectrophoretic highfield region performed in fluid conductivity of less than about 300mS/m. In some embodiments, the DEP field region is a dielectrophoreticlow field region performed in fluid conductivity of greater than about500 mS/m. In some embodiments, the DEP field region is adielectrophoretic low field region performed in fluid conductivity ofless than about 500 mS/m. In some embodiments, the DEP field region is adielectrophoretic high field region performed in fluid conductivity ofgreater than about 500 mS/m. In some embodiments, the DEP field regionis a dielectrophoretic high field region performed in fluid conductivityof less than about 500 mS/m.

In some embodiments, the dielectrophoretic field region is produced byan alternating current. The alternating current has any amperage,voltage, frequency, and the like suitable for concentrating cells. Insome embodiments, the dielectrophoretic field region is produced usingan alternating current having an amperage of 0.1 micro Amperes-10Amperes; a voltage of 1-50 Volts peak to peak; and/or a frequency of1-10,000,000 Hz. In some embodiments, the DEP field region is producedusing an alternating current having a voltage of 5-25 volts peak topeak. In some embodiments, the DEP field region is produced using analternating current having a frequency of from 3-15 kHz.

In some embodiments, the DEP field region is produced using analternating current having an amperage of 100 milliamps to 5 amps. Insome embodiments, the DEP field region is produced using an alternatingcurrent having an amperage of 0.5 Ampere-1 Ampere. In some embodiments,the DEP field region is produced using an alternating current having anamperage of 0.5 Ampere-5 Ampere. In some embodiments, the DEP fieldregion is produced using an alternating current having an amperage of100 milliamps-1 Ampere. In some embodiments, the DEP field region isproduced using an alternating current having an amperage of 500 milliAmperes-2.5 Amperes.

In some embodiments, the DEP field region is produced using analternating current having a voltage of 1-25 Volts peak to peak. In someembodiments, the DEP field region is produced using an alternatingcurrent having a voltage of 1-10 Volts peak to peak. In someembodiments, the DEP field region is produced using an alternatingcurrent having a voltage of 25-50 Volts peak to peak. In someembodiments, the DEP field region is produced using a frequency of from10-1,000,000 Hz. In some embodiments, the DEP field region is producedusing a frequency of from 100-100,000 Hz. In some embodiments, the DEPfield region is produced using a frequency of from 100-10,000 Hz. Insome embodiments, the DEP field region is produced using a frequency offrom 10,000-100,000 Hz. In some embodiments, the DEP field region isproduced using a frequency of from 100,000-1,000,000 Hz.

In some embodiments, the first dielectrophoretic field region isproduced by a direct current. The direct current has any amperage,voltage, frequency, and the like suitable for concentrating cells. Insome embodiments, the first dielectrophoretic field region is producedusing a direct current having an amperage of 0.1 micro Amperes-1Amperes; a voltage of 10 milli Volts-10 Volts; and/or a pulse width of 1milliseconds-1000 seconds and a pulse frequency of 0.001-1000 Hz. Insome embodiments, the DEP field region is produced using a directcurrent having an amperage of 1 micro Amperes-1 Amperes. In someembodiments, the DEP field region is produced using a direct currenthaving an amperage of 100 micro Amperes-500 milli Amperes. In someembodiments, the DEP field region is produced using a direct currenthaving an amperage of 1 milli Amperes-1 Amperes. In some embodiments,the DEP field region is produced using a direct current having anamperage of 1 micro Amperes-1 milli Amperes. In some embodiments, theDEP field region is produced using a direct current having a pulse widthof 500 milliseconds-500 seconds. In some embodiments, the DEP fieldregion is produced using a direct current having a pulse width of 500milliseconds-100 seconds. In some embodiments, the DEP field region isproduced using a direct current having a pulse width of 1 second-1000seconds. In some embodiments, the DEP field region is produced using adirect current having a pulse width of 500 milliseconds-1 second. Insome embodiments, the DEP field region is produced using a pulsefrequency of 0.01-1000 Hz. In some embodiments, the DEP field region isproduced using a pulse frequency of 0.1-100 Hz. In some embodiments, theDEP field region is produced using a pulse frequency of 1-100 Hz. Insome embodiments, the DEP field region is produced using a pulsefrequency of 100-1000 Hz.

In some embodiments, the sample may comprise a mixture of cell types.For example, blood comprises red blood cells and white blood cells.Environmental samples comprise many types of cells and other particulatematerial over a wide range of concentrations. In some embodiments, onecell type (or any number of cell types less than the total number ofcell types comprising the sample) may be preferentially concentrated ina DEP field region. In another non-limiting example, the DEP field isoperated in a manner that specifically concentrates viruses and notcells (e.g., in a fluid with conductivity of greater than 300 mS/m,viruses concentrate in a DEP high field region, while larger cells willconcentrate in a DEP low field region).

Accordingly, in some embodiments, a method, device or system describedherein is suitable for isolating or separating specific cell types inorder to enable efficient isolation and collection of nanoscaleanalytes. In some embodiments, the DEP field of the method, device orsystem is specifically tuned to allow for the separation orconcentration of a specific type of cell into a field region of the DEPfield. In some embodiments, a method, device or system described hereinprovides more than one field region wherein more than one type of cellis isolated or concentrated. In some embodiments, a method, device, orsystem described herein is tunable so as to allow isolation orconcentration of different types of cells within the DEP field regionsthereof. In some embodiments, a method provided herein further comprisestuning the DEP field. In some embodiments, a device or system providedherein is capable of having the DEP field tuned. In some instances, suchtuning may be in providing a DEP particularly suited for the desiredpurpose. For example, modifications in the array, the energy, or anotherparameter are optionally utilized to tune the DEP field. Tuningparameters for finer resolution include electrode diameter, edge to edgedistance between electrodes, voltage, frequency, fluid conductivity andhydrogel composition.

In some embodiments, the DEP field region comprises the entirety of anarray of electrodes as disclosed herein. In some embodiments, the DEPfield region comprises a portion of an array of electrodes as disclosedherein. In some embodiments, the DEP field region comprises about 90%,about 80%, about 70%, about 60%, about 50%, about 40%, about 30%, about25%, about 20%, or about 10% of an array of electrodes as disclosedherein. In some embodiments, the DEP field region comprises about athird of an array of electrodes as disclosed herein.

Cell Lysis

In one aspect, following concentrating the cells in a firstdielectrophoretic field region, the method involves freeing nanoscaleanalytes from the cell. In another aspect, the devices and systemsdescribed herein are capable of freeing nucleic acids from the cells. Insome embodiments, the nucleic acids are freed from the cells in thefirst DEP field region.

In some embodiments, the methods described herein free nucleic acidsfrom a plurality of cells by lysing the cells. In some embodiments, thedevices and systems described herein are capable of freeing nucleicacids from a plurality of cells by lysing the cells. One method of celllysis involves applying a direct current to the cells after isolation ofthe cells on the array. The direct current has any suitable amperage,voltage, and the like suitable for lysing cells. In some embodiments,the current has a voltage of about 1 Volt to about 500 Volts. In someembodiments, the current has a voltage of about 10 Volts to about 500Volts. In other embodiments, the current has a voltage of about 10 Voltsto about 250 Volts. In still other embodiments, the current has avoltage of about 50 Volts to about 150 Volts. Voltage is generally thedriver of cell lysis, as high electric fields result in failed membraneintegrity.

In some embodiments, the direct current used for lysis comprises one ormore pulses having any duration, frequency, and the like suitable forlysing cells. In some embodiments, a voltage of about 100 volts isapplied for about 1 millisecond to lyse cells. In some embodiments, thevoltage of about 100 volts is applied 2 or 3 times over the source of asecond.

In some embodiments, the frequency of the direct current depends onvolts/cm, pulse width, and the fluid conductivity. In some embodiments,the pulse has a frequency of about 0.001 to about 1000 Hz. In someembodiments, the pulse has a frequency from about 10 to about 200 Hz. Inother embodiments, the pulse has a frequency of about 0.01 Hz-1000 Hz.In still other embodiments, the pulse has a frequency of about 0.1Hz-1000 Hz, about 1 Hz-1000 Hz, about 1 Hz-500 Hz, about 1 Hz-400 Hz,about 1 Hz-300 Hz, or about 1 Hz-about 250 Hz. In some embodiments, thepulse has a frequency of about 0.1 Hz. In other embodiments, the pulsehas a frequency of about 1 Hz. In still other embodiments, the pulse hasa frequency of about 5 Hz, about 10 Hz, about 50 Hz, about 100 Hz, about200 Hz, about 300 Hz, about 400 Hz, about 500 Hz, about 600 Hz, about700 Hz, about 800 Hz, about 900 Hz or about 1000 Hz.

In other embodiments, the pulse has a duration of about 1 millisecond(ms)-1000 seconds (s). In some embodiments, the pulse has a duration ofabout 10 ms-1000 s. In still other embodiments, the pulse has a durationof about 100 ms-1000 s, about 1 s-1000 s, about 1 s-500 s, about 1 s-250s or about 1 s-150 s. In some embodiments, the pulse has a duration ofabout 1 ms, about 10 ms, about 100 ms, about 1 s, about 2 s, about 3 s,about 4 s, about 5 s, about 6 s, about 7 s, about 8 s, about 9 s, about10 s, about 20 s, about 50 s, about 100 s, about 200 s, about 300 s,about 500 s or about 1000 s. In some embodiments, the pulse has afrequency of 0.2 to 200 Hz with duty cycles from 10-50%.

In some embodiments, the direct current is applied once, or as multiplepulses. Any suitable number of pulses may be applied including about1-20 pulses. There is any suitable amount of time between pulsesincluding about 1 millisecond-1000 seconds. In some embodiments, thepulse duration is 0.01 to 10 seconds.

In some embodiments, the cells are lysed using other methods incombination with a direct current applied to the isolated cells. In yetother embodiments, the cells are lysed without use of direct current. Invarious aspects, the devices and systems are capable of lysing cellswith direct current in combination with other means, or may be capableof lysing cells without the use of direct current. Any method of celllysis known to those skilled in the art may be suitable including, butnot limited to application of a chemical lysing agent (e.g., an acid),an enzymatic lysing agent, heat, pressure, shear force, sonic energy,osmotic shock, or combinations thereof. Lysozyme is an example of anenzymatic-lysing agent.

Nanoscale Analytes Isolation and Yields Thereof

In one aspect, described herein are methods and devices for isolating ananoscale analyte from a sample. In some embodiments, the nanoscaleanalyte is less than 1000 nm in diameter. In other embodiments, thenanoscale analyte is less than 500 nm in diameter. In some embodiments,the nanoscale analyte is less than 250 nm in diameter. In someembodiments, the nanoscale analyte is between about 100 nm to about 1000nm in diameter. In other embodiments, the nanoscale analyte is betweenabout 250 nm to about 800 nm in diameter. In still other embodiments,the nanoscale analyte is between about 300 nm to about 500 nm indiameter.

In some embodiments, the nanoscale analyte is less than 1000 μm indiameter. In other embodiments, the nanoscale analyte is less than 500μm in diameter. In some embodiments, the nanoscale analyte is less than250 μm in diameter. In some embodiments, the nanoscale analyte isbetween about 100 μm to about 1000 μm in diameter. In other embodiments,the nanoscale analyte is between about 250 μm to about 800 μm indiameter. In still other embodiments, the nanoscale analyte is betweenabout 300 μm to about 500 μm in diameter.

In some embodiments, the method, device, or system described herein isoptionally utilized to obtain, isolate, or separate any desirednanoscale analyte that may be obtained from such a method, device orsystem. In some embodiments, the nanoscale analyte is a nucleic acid. Inother the nucleic acids isolated by the methods, devices and systemsdescribed herein include DNA (deoxyribonucleic acid), RNA (ribonucleicacid), and combinations thereof. In some embodiments, the nucleic acidis isolated in a form suitable for sequencing or further manipulation ofthe nucleic acid, including amplification, ligation or cloning.

In various embodiments, an isolated or separated nanoscale analyte is acomposition comprising nanoscale analyte that is free from at least 99%by mass of other materials, free from at least 99% by mass of residualcellular material, free from at least 98% by mass of other materials,free from at least 98% by mass of residual cellular material, free fromat least 95% by mass of other materials, free from at least 95% by massof residual cellular material, free from at least 90% by mass of othermaterials, free from at least 90% by mass of residual cellular material,free from at least 80% by mass of other materials, free from at least80% by mass of residual cellular material, free from at least 70% bymass of other materials, free from at least 70% by mass of residualcellular material, free from at least 60% by mass of other materials,free from at least 60% by mass of residual cellular material, free fromat least 50% by mass of other materials, free from at least 50% by massof residual cellular material, free from at least 30% by mass of othermaterials, free from at least 30% by mass of residual cellular material,free from at least 10% by mass of other materials, free from at least10% by mass of residual cellular material, free from at least 5% by massof other materials, or free from at least 5% by mass of residualcellular material.

In various embodiments, the nanoscale analyte has any suitable purity.For example, if a enzymatic assay requires nanoscale analyte sampleshaving about 20% residual cellular material, then isolation of thenucleic acid to 80% is suitable. In some embodiments, the isolatednanoscale analyte comprises less than about 80%, less than about 70%,less than about 60%, less than about 50%, less than about 40%, less thanabout 30%, less than about 20%, less than about 10%, less than about 5%,or less than about 2% non-nanoscale analyte cellular material and/orprotein by mass. In some embodiments, the isolated nanoscale analytecomprises greater than about 99%, greater than about 98%, greater thanabout 95%, greater than about 90%, greater than about 80%, greater thanabout 70%, greater than about 60%, greater than about 50%, greater thanabout 40%, greater than about 30%, greater than about 20%, or greaterthan about 10% nanoscale analyte by mass.

The nanoscale analytes are isolated in any suitable form includingunmodified, derivatized, fragmented, non-fragmented, and the like. Insome embodiments, when the nanoscale analyte is a nucleic acid, thenucleic acid is collected in a form suitable for sequencing. In someembodiments, the nucleic acid is collected in a fragmented form suitablefor shotgun-sequencing, amplification or other manipulation. The nucleicacid may be collected from the device in a solution comprising reagentsused in, for example, a DNA sequencing procedure, such as nucleotides asused in sequencing by synthesis methods.

In some embodiments, the methods described herein result in an isolatednanoscale analyte sample that is approximately representative of thenanoscale analyte of the starting sample. In some embodiments, thedevices and systems described herein are capable of isolating nanoscaleanalyte from a sample that is approximately representative of thenanoscale analyte of the starting sample. That is, the population ofnanoscale analytes collected by the method, or capable of beingcollected by the device or system, are substantially in proportion tothe population of nanoscale analytes present in the cells in the fluid.In some embodiments, this aspect is advantageous in applications inwhich the fluid is a complex mixture of many cell types and thepractitioner desires a nanoscale analyte-based procedure for determiningthe relative populations of the various cell types.

In some embodiments, the nanoscale analyte isolated by the methodsdescribed herein or capable of being isolated by the devices describedherein has a concentration of at least 0.5 ng/mL. In some embodiments,the nanoscale analyte isolated by the methods described herein orcapable of being isolated by the devices described herein has aconcentration of at least 1 ng/mL. In some embodiments, the nanoscaleanalyte isolated by the methods described herein or capable of beingisolated by the devices described herein has a concentration of at least5 ng/mL. In some embodiments, the nanoscale analyte isolated by themethods described herein or capable of being isolated by the devicesdescribed herein has a concentration of at least 10 ng/ml.

In some embodiments, about 50 pico-grams of nanoscale analyte isisolated from a sample comprising about 5,000 cells using the methods,systems or devices described herein. In some embodiments, the methods,systems or devices described herein yield at least 10 pico-grams ofnanoscale analyte from a sample comprising about 5,000 cells. In someembodiments, the methods, systems or devices described herein yield atleast 20 pico-grams of nanoscale analyte from a sample comprising about5,000 cells. In some embodiments, the methods, systems or devicesdescribed herein yield at least 50 pico-grams of nanoscale analyte fromabout 5,000 cells. In some embodiments, the methods, systems or devicesdescribed herein yield at least 75 pico-grams of nanoscale analyte froma sample comprising about 5,000 cells. In some embodiments, the methods,systems or devices described herein yield at least 100 pico-grams ofnanoscale analyte from a sample comprising about 5,000 cells. In someembodiments, the methods, systems or devices described herein yield atleast 200 pico-grams of nanoscale analyte from a sample comprising about5,000 cells. In some embodiments, the methods, systems or devicesdescribed herein yield at least 300 pico-grams of nanoscale analyte froma sample comprising about 5,000 cells. In some embodiments, the methods,systems or devices described herein yield at least 400 pico-grams ofnanoscale analyte from a sample comprising about 5,000 cells. In someembodiments, the methods, systems or devices described herein yield atleast 500 pico-grams of nanoscale analyte from a sample comprising about5,000 cells. In some embodiments, the methods, systems or devicesdescribed herein yield at least 1,000 pico-grams of nanoscale analytefrom a sample comprising about 5,000 cells. In some embodiments, themethods, systems or devices described herein yield at least 10,000pico-grams of nanoscale analyte from a sample comprising about 5,000cells. In some embodiments, the methods, systems or devices describedherein yield at least 20,000 pico-grams of nanoscale analyte from asample comprising about 5,000 cells. In some embodiments, the methods,systems or devices described herein yield at least 30,000 pico-grams ofnanoscale analyte from a sample comprising about 5,000 cells. In someembodiments, the methods, systems or devices described herein yield atleast 40,000 pico-grams of nanoscale analyte from a sample comprisingabout 5,000 cells. In some embodiments, the methods, systems or devicesdescribed herein yield at least 50,000 pico-grams of nanoscale analytefrom a sample comprising about 5,000 cells.

When the nanoscale analyte is a nucleic acid, the nucleic acid isolatedusing the methods described herein or capable of being isolated by thedevices described herein is high-quality and/or suitable for usingdirectly in downstream procedures such as DNA sequencing, nucleic acidamplification, such as PCR, or other nucleic acid manipulation, such asligation, cloning or further translation or transformation assays. Insome embodiments, the collected nucleic acid comprises at most 0.01%protein. In some embodiments, the collected nucleic acid comprises atmost 0.5% protein. In some embodiments, the collected nucleic acidcomprises at most 0.1% protein. In some embodiments, the collectednucleic acid comprises at most 1% protein. In some embodiments, thecollected nucleic acid comprises at most 2% protein. In someembodiments, the collected nucleic acid comprises at most 3% protein. Insome embodiments, the collected nucleic acid comprises at most 4%protein. In some embodiments, the collected nucleic acid comprises atmost 5% protein.

Samples

In one aspect, the methods, systems and devices described herein isolatenanoscale analytes from a sample. In some embodiments, the samplecomprises a fluid. In one aspect, the sample comprises cells or otherparticulate material and the nanoscale analytes. In some embodiments,the sample does not comprise cells.

In some embodiments, the sample is a liquid, optionally water or anaqueous solution or dispersion. In some embodiments, the sample is abodily fluid. Exemplary bodily fluids include blood, serum, plasma,bile, milk, cerebrospinal fluid, gastric juice, ejaculate, mucus,peritoneal fluid, saliva, sweat, tears, urine, synovial fluid and thelike. In some embodiments, nanoscale analytes are isolated from bodilyfluids using the methods, systems or devices described herein as part ofa medical therapeutic or diagnostic procedure, device or system. In someembodiments, the sample is tissues and/or cells solubilized and/ordispersed in a fluid medium. For example, the tissue can be a canceroustumor from which nanoscale analytes, such as nucleic acids, can beisolated using the methods, devices or systems described herein.

In some embodiments, the sample is an environmental sample. In someembodiments, the environmental sample is assayed or monitored for thepresence of a particular nucleic acid sequence indicative of a certaincontamination, infestation incidence or the like. The environmentalsample can also be used to determine the source of a certaincontamination, infestation incidence or the like using the methods,devices or systems described herein. Exemplary environmental samplesinclude municipal wastewater, industrial wastewater, water or fluid usedin or produced as a result of various manufacturing processes, lakes,rivers, oceans, aquifers, ground water, storm water, plants or portionsof plants, animals or portions of animals, insects, municipal watersupplies, and the like.

In some embodiments, the sample is a food or beverage. The food orbeverage can be assayed or monitored for the presence of a particularnanoscale analyte indicative of a certain contamination, infestationincidence or the like. The food or beverage can also be used todetermine the source of a certain contamination, infestation incidenceor the like using the methods, devices or systems described herein. Invarious embodiments, the methods, devices and systems described hereincan be used with one or more of bodily fluids, environmental samples,and foods and beverages to monitor public health or respond to adversepublic health incidences.

In some embodiments, the sample is a growth medium. The growth mediumcan be any medium suitable for culturing cells, for example lysogenybroth (LB) for culturing E. coli, Ham's tissue culture medium forculturing mammalian cells, and the like. The medium can be a richmedium, minimal medium, selective medium, and the like. In someembodiments, the medium comprises or consists essentially of a pluralityof clonal cells. In some embodiments, the medium comprises a mixture ofat least two species.

In some embodiments, the sample is water.

In some embodiments, the sample may also comprise other particulatematerial. Such particulate material may be, for example, inclusionbodies (e.g., ceroids or Mallory bodies), cellular casts (e.g., granularcasts, hyaline casts, cellular casts, waxy casts and pseudo casts),Pick's bodies, Lewy bodies, fibrillary tangles, fibril formations,cellular debris and other particulate material. In some embodiments,particulate material is an aggregated protein (e.g., beta-amyloid).

The sample can have any conductivity including a high or lowconductivity. In some embodiments, the conductivity is between about 1μS/m to about 10 mS/m. In some embodiments, the conductivity is betweenabout 10 μS/m to about 10 mS/m. In other embodiments, the conductivityis between about 50 μS/m to about 10 mS/m. In yet other embodiments, theconductivity is between about 100 μS/m to about 10 mS/m, between about100 μS/m to about 8 mS/m, between about 100 μS/m to about 6 mS/m,between about 100 μS/m to about 5 mS/m, between about 100 μS/m to about4 mS/m, between about 100 μS/m to about 3 mS/m, between about 100 μS/mto about 2 mS/m, or between about 100 μS/m to about 1 mS/m.

In some embodiments, the conductivity is about 1 μS/m. In someembodiments, the conductivity is about 10 μS/m. In some embodiments, theconductivity is about 100 μS/m. In some embodiments, the conductivity isabout 1 mS/m. In other embodiments, the conductivity is about 2 mS/m. Insome embodiments, the conductivity is about 3 mS/m. In yet otherembodiments, the conductivity is about 4 mS/m. In some embodiments, theconductivity is about 5 mS/m. In some embodiments, the conductivity isabout 10 mS/m. In still other embodiments, the conductivity is about 100mS/m. In some embodiments, the conductivity is about 1 S/m. In otherembodiments, the conductivity is about 10 S/m.

In some embodiments, the conductivity is at least 1 μS/m. In yet otherembodiments, the conductivity is at least 10 μS/m. In some embodiments,the conductivity is at least 100 μS/m. In some embodiments, theconductivity is at least 1 mS/m. In additional embodiments, theconductivity is at least 10 mS/m. In yet other embodiments, theconductivity is at least 100 mS/m. In some embodiments, the conductivityis at least 1 S/m. In some embodiments, the conductivity is at least 10S/m. In some embodiments, the conductivity is at most 1 μS/m. In someembodiments, the conductivity is at most 10 μS/m. In other embodiments,the conductivity is at most 100 μS/m. In some embodiments, theconductivity is at most 1 mS/m. In some embodiments, the conductivity isat most 10 mS/m. In some embodiments, the conductivity is at most 100mS/m. In yet other embodiments, the conductivity is at most 1 S/m. Insome embodiments, the conductivity is at most 10 S/m.

In some embodiments, the sample is a small volume of liquid includingless than 10 ml. In some embodiments, the sample is less than 8 ml. Insome embodiments, the sample is less than 5 ml. In some embodiments, thesample is less than 2 ml. In some embodiments, the sample is less than 1ml. In some embodiments, the sample is less than 500 μl. In someembodiments, the sample is less than 200 μl. In some embodiments, thesample is less than 100 μl. In some embodiments, the sample is less than50 μl. In some embodiments, the sample is less than 10 μl. In someembodiments, the sample is less than 5 μl. In some embodiments, thesample is less than 1 μl.

In some embodiments, the quantity of sample applied to the device orused in the method comprises less than about 100,000,000 cells. In someembodiments, the sample comprises less than about 10,000,000 cells. Insome embodiments, the sample comprises less than about 1,000,000 cells.In some embodiments, the sample comprises less than about 100,000 cells.In some embodiments, the sample comprises less than about 10,000 cells.In some embodiments, the sample comprises less than about 1,000 cells.

In some embodiments, isolation of a nanoscale analyte from a sample withthe devices, systems and methods described herein takes less than about30 minutes, less than about 20 minutes, less than about 15 minutes, lessthan about 10 minutes, less than about 5 minutes or less than about 1minute. In other embodiments, isolation of a nanoscale analyte from asample with the devices, systems and methods described herein takes notmore than 30 minutes, not more than about 20 minutes, not more thanabout 15 minutes, not more than about 10 minutes, not more than about 5minutes, not more than about 2 minutes or not more than about 1 minute.In additional embodiments, isolation of a nanoscale analyte from asample with the devices, systems and methods described herein takes lessthan about 15 minutes, preferably less than about 10 minutes or lessthan about 5 minutes.

Removal of Residual Material

In some embodiments, following isolation of the nanoscale analytes in aDEP field region, the method includes optionally flushing residualmaterial from the isolated nanoscale analytes. In some embodiments, thedevices or systems described herein are capable of optionally and/orcomprising a reservoir comprising a fluid suitable for flushing residualmaterial from the nanoscale analytes. “Residual material” is anythingoriginally present in the sample, originally present in the cells, addedduring the procedure, created through any step of the process includingbut not limited to cells (e.g. intact cells or residual cellularmaterial), and the like. For example, residual material includes intactcells, cell wall fragments, proteins, lipids, carbohydrates, minerals,salts, buffers, plasma, and the like. In some embodiments, a certainamount of nanoscale analyte is flushed with the residual material.

In some embodiments, the residual material is flushed in any suitablefluid, for example in water, TBE buffer, or the like. In someembodiments, the residual material is flushed with any suitable volumeof fluid, flushed for any suitable period of time, flushed with morethan one fluid, or any other variation. In some embodiments, the methodof flushing residual material is related to the desired level ofisolation of the nanoscale analyte, with higher purity nanoscale analyterequiring more stringent flushing and/or washing. In other embodiments,the method of flushing residual material is related to the particularstarting material and its composition. In some instances, a startingmaterial that is high in lipid requires a flushing procedure thatinvolves a hydrophobic fluid suitable for solubilizing lipids.

In some embodiments, the method includes degrading residual materialincluding residual protein. In some embodiments, the devices or systemsare capable of degrading residual material including residual protein.For example, proteins are degraded by one or more of chemicaldegradation (e.g. acid hydrolysis) and enzymatic degradation. In someembodiments, the enzymatic degradation agent is a protease. In otherembodiments, the protein degradation agent is Proteinase K. The optionalstep of degradation of residual material is performed for any suitabletime, temperature, and the like. In some embodiments, the degradedresidual material (including degraded proteins) is flushed from theisolated nanoscale analytes.

In some embodiments, the agent used to degrade the residual material isinactivated or degraded. In some embodiments, the devices or systems arecapable of degrading or inactivating the agent used to degrade theresidual material. In some embodiments, an enzyme used to degrade theresidual material is inactivated by heat (e.g., 50 to 95° C. for 5-15minutes). For example, enzymes including proteases, (for example,Proteinase K) are degraded and/or inactivated using heat (typically, 15minutes, 70° C.). In some embodiments wherein the residual proteins aredegraded by an enzyme, the method further comprises inactivating thedegrading enzyme (e.g., Proteinase K) following degradation of theproteins. In some embodiments, heat is provided by a heating module inthe device (temperature range, e.g., from 30 to 95° C.).

The order and/or combination of certain steps of the method can bevaried. In some embodiments, the devices or methods are capable ofperforming certain steps in any order or combination. For example, insome embodiments, the residual material and the degraded proteins areflushed in separate or concurrent steps. That is, the residual materialis flushed, followed by degradation of residual proteins, followed byflushing degraded proteins from the isolated nanoscale analytes. In someembodiments, one first degrades the residual proteins, and then flushboth the residual material and degraded proteins from the nanoscaleanalytes in a combined step.

In some embodiments, the nanoscale analytes are retained in the deviceand optionally used in further procedures, such as PCR, enzymatic assaysor other procedures that analyze, characterize or amplify the nanoscaleanalytes.

For example, in some embodiments, the isolated nanoscale analyte is anucleic acid, and the devices and systems are capable of performing PCRor other optional procedures on the isolated nucleic acids. In otherembodiments, the nucleic acids are collected and/or eluted from thedevice. In some embodiments, the devices and systems are capable ofallowing collection and/or elution of nucleic acid from the device orsystem. In some embodiments, the isolated nucleic acid is collected by(i) turning off the second dielectrophoretic field region; and (ii)eluting the nucleic acid from the array in an eluant. Exemplary eluantsinclude water, TE, TBE and L-Histidine buffer.

Assays and Applications

In some embodiments, a system or device described herein includes ameans of performing enzymatic reactions. In other embodiments, a systemor device described herein includes a means of performing polymerasechain reaction (PCR), isothermal amplification, ligation reactions,restriction analysis, nucleic acid cloning, transcription or translationassays, or other enzymatic-based molecular biology assay.

In some embodiments, a system or device described herein comprises anucleic acid sequencer. The sequencer is optionally any suitable DNAsequencing device including but not limited to a Sanger sequencer,pyro-sequencer, ion semiconductor sequencer, polony sequencer,sequencing by ligation device, DNA nanoball sequencing device,sequencing by ligation device, or single molecule sequencing device.

In some embodiments, the methods described herein further compriseoptionally amplifying the isolated nucleic acid by polymerase chainreaction (PCR). In some embodiments, the PCR reaction is performed on ornear the array of electrodes or in the device. In some embodiments, thedevice or system comprise a heater and/or temperature control mechanismssuitable for thermocycling.

PCR is optionally done using traditional thermocycling by placing thereaction chemistry analytes in between two efficient thermoconductiveelements (e.g., aluminum or silver) and regulating the reactiontemperatures using TECs. Additional designs optionally use infraredheating through optically transparent material like glass or thermopolymers. In some instances, designs use smart polymers or smart glassthat comprise conductive wiring networked through the substrate. Thisconductive wiring enables rapid thermal conductivity of the materialsand (by applying appropriate DC voltage) provides the requiredtemperature changes and gradients to sustain efficient PCR reactions. Incertain instances, heating is applied using resistive chip heaters andother resistive elements that will change temperature rapidly andproportionally to the amount of current passing through them.

In some embodiments, used in conjunction with traditional fluorometry(ccd, pmt, other optical detector, and optical filters), foldamplification is monitored in real-time or on a timed interval. Incertain instances, quantification of final fold amplification isreported via optical detection converted to AFU (arbitrary fluorescenceunits correlated to analyze doubling) or translated to electrical signalvia impedance measurement or other electrochemical sensing.

Given the small size of the micro electrode array, these elements areoptionally added around the micro electrode array and the PCR reactionwill be performed in the main sample processing chamber (over the DEParray) or the analytes to be amplified are optionally transported viafluidics to another chamber within the fluidic cartridge to enableon-cartridge Lab-On-Chip processing.

In some instances, light delivery schemes are utilized to provide theoptical excitation and/or emission and/or detection of foldamplification. In certain embodiments, this includes using the flow cellmaterials (thermal polymers like acrylic (PMMA) cyclic olefin polymer(COP), cyclic olefin co-polymer, (COC), etc.) as optical wave guides toremove the need to use external components. In addition, in someinstances light sources—light emitting diodes—LEDs, vertical-cavitysurface-emitting lasers—VCSELs, and other lighting schemes areintegrated directly inside the flow cell or built directly onto themicro electrode array surface to have internally controlled and poweredlight sources. Miniature PMTs, CCDs, or CMOS detectors can also be builtinto the flow cell. This minimization and miniaturization enablescompact devices capable of rapid signal delivery and detection whilereducing the footprint of similar traditional devices (i.e. a standardbench top PCR/QPCR/Fluorometer).

Amplification on Chip

In some instances, silicon microelectrode arrays can withstand thermalcycling necessary for PCR. In some applications, on-chip PCR isadvantageous because small amounts of target nucleic acids can be lostduring transfer steps. In certain embodiments of devices, systems orprocesses described herein, any one or more of multiple PCR techniquesare optionally used, such techniques optionally including any one ormore of the following: thermal cycling in the flowcell directly; movingthe material through microchannels with different temperature zones; andmoving volume into a PCR tube that can be amplified on system ortransferred to a PCR machine. In some instances, droplet PCR isperformed if the outlet contains a T-junction that contains animmiscible fluid and interfacial stabilizers (surfactants, etc). Incertain embodiments, droplets are thermal cycled in by any suitablemethod.

In some embodiments, amplification is performed using an isothermalreaction, for example, transcription mediated amplification, nucleicacid sequence-based amplification, signal mediated amplification of RNAtechnology, strand displacement amplification, rolling circleamplification, loop-mediated isothermal amplification of DNA, isothermalmultiple displacement amplification, helicase-dependent amplification,single primer isothermal amplification or circular helicase-dependentamplification.

In various embodiments, amplification is performed in homogenoussolution or as heterogeneous system with anchored primer(s). In someembodiments of the latter, the resulting amplicons are directly linkedto the surface for higher degree of multiplex. In some embodiments, theamplicon is denatured to render single stranded products on or near theelectrodes. Hybridization reactions are then optionally performed tointerrogate the genetic information, such as single nucleotidepolymorphisms (SNPs), Short Tandem Repeats (STRs), mutations,insertions/deletions, methylation, etc. Methylation is optionallydetermined by parallel analysis where one DNA sample is bisulfitetreated and one is not. Bisulfite depurinates unmodified C becoming a U.Methylated C is unaffected in some instances. In some embodiments,allele specific base extension is used to report the base of interest.

Rather than specific interactions, the surface is optionally modifiedwith nonspecific moieties for capture. For example, surface could bemodified with polycations, i.e., polylysine, to capture DNA moleculeswhich can be released by reverse bias (−V). In some embodiments,modifications to the surface are uniform over the surface or patternedspecifically for functionalizing the electrodes or non electroderegions. In certain embodiments, this is accomplished withphotolithography, electrochemical activation, spotting, and the like.

In some applications, where multiple chip designs are employed, it isadvantageous to have a chip sandwich where the two devices are facingeach other, separated by a spacer, to form the flow cell. In variousembodiments, devices are run sequentially or in parallel. For sequencingand next generation sequencing (NGS), size fragmentation and selectionhas ramifications on sequencing efficiency and quality. In someembodiments, multiple chip designs are used to narrow the size range ofmaterial collected creating a band pass filter. In some instances,current chip geometry (e.g., 80 μm diameter electrodes on 200 μmcenter-center pitch (80/200) acts as 500 bp cutoff filter (e.g., usingvoltage and frequency conditions around 10 Vpp and 10 kHz). In suchinstances, a nucleic acid of greater than 500 bp is captured, and anucleic acid of less than 500 bp is not. Alternate electrode diameterand pitch geometries have different cutoff sizes such that a combinationof chips should provide a desired fragment size. In some instances, a 40μm diameter electrode on 100 μm center-center pitch (40/100) has a lowercutoff threshold, whereas a 160 μm diameter electrode on 400 μmcenter-center pitch (160/400) has a higher cutoff threshold relative tothe 80/200 geometry, under similar conditions. In various embodiments,geometries on a single chip or multiple chips are combined to select fora specific sized fragments or particles. For example a 600 bp cutoffchip would leave a nucleic acid of less than 600 bp in solution, thenthat material is optionally recaptured with a 500 bp cutoff chip (whichis opposing the 600 bp chip). This leaves a nucleic acid populationcomprising 500-600 bp in solution. This population is then optionallyamplified in the same chamber, a side chamber, or any otherconfiguration. In some embodiments, size selection is accomplished usinga single electrode geometry, wherein nucleic acid of >500 bp is isolatedon the electrodes, followed by washing, followed by reduction of theACEK high field strength (change voltage, frequency, conductivity) inorder to release nucleic acids of <600 bp, resulting in a supernatantnucleic acid population between 500-600 bp.

In some embodiments, the chip device is oriented vertically with aheater at the bottom edge which creates a temperature gradient column.In certain instances, the bottom is at denaturing temperature, themiddle at annealing temperature, the top at extension temperature. Insome instances, convection continually drives the process. In someembodiments, provided herein are methods or systems comprising anelectrode design that specifically provides for electrothermal flows andacceleration of the process. In some embodiments, such design isoptionally on the same device or on a separate device positionedappropriately. In some instances, active or passive cooling at the top,via fins or fans, or the like provides a steep temperature gradient. Insome instances the device or system described herein comprises, or amethod described herein uses, temperature sensors on the device or inthe reaction chamber monitor temperature and such sensors are optionallyused to adjust temperature on a feedback basis. In some instances, suchsensors are coupled with materials possessing different thermal transferproperties to create continuous and/or discontinuous gradient profiles.

In some embodiments, the amplification proceeds at a constanttemperature (i.e, isothermal amplification).

In some embodiments, the methods disclosed herein further comprisesequencing the nucleic acid isolated as disclosed herein. In someembodiments, the nucleic acid is sequenced by Sanger sequencing or nextgeneration sequencing (NGS). In some embodiments, the next generationsequencing methods include, but are not limited to, pyrosequencing, ionsemiconductor sequencing, polony sequencing, sequencing by ligation, DNAnanoball sequencing, sequencing by ligation, or single moleculesequencing.

In some embodiments, the isolated nucleic acids disclosed herein areused in Sanger sequencing. In some embodiments, Sanger sequencing isperformed within the same device as the nucleic acid isolation(Lab-on-Chip). Lab-on-Chip workflow for sample prep and Sangersequencing results would incorporate the following steps: a) sampleextraction using ACE chips; b) performing amplification of targetsequences on chip; c) capture PCR products by ACE; d) perform cyclesequencing to enrich target strand; e) capture enriched target strands;f) perform Sanger chain termination reactions; perform electrophoreticseparation of target sequences by capillary electrophoresis with on chipmulti-color fluorescence detection. Washing nucleic acids, addingreagent, and turning off voltage is performed as necessary. Reactionscan be performed on a single chip with plurality of capture zones or onseparate chips and/or reaction chambers.

In some embodiments, the method disclosed herein further compriseperforming a reaction on the nucleic acids (e.g., fragmentation,restriction digestion, ligation of DNA or RNA). In some embodiments, thereaction occurs on or near the array or in a device, as disclosedherein.

Other Assays

The isolated nucleic acids disclosed herein may be further utilized in avariety of assay formats. For instance, devices which are addressed withnucleic acid probes or amplicons may be utilized in dot blot or reversedot blot analyses, base-stacking single nucleotide polymorphism (SNP)analysis, SNP analysis with electronic stringency, or in STR analysis.In addition, such devices disclosed herein may be utilized in formatsfor enzymatic nucleic acid modification, or protein-nucleic acidinteraction, such as, e.g., gene expression analysis with enzymaticreporting, anchored nucleic acid amplification, or other nucleic acidmodifications suitable for solid-phase formats including restrictionendonuclease cleavage, endo- or exo-nuclease cleavage, minor groovebinding protein assays, terminal transferase reactions, polynucleotidekinase or phosphatase reactions, ligase reactions, topoisomerasereactions, and other nucleic acid binding or modifying proteinreactions.

In addition, the devices disclosed herein can be useful in immunoassays.For instance, in some embodiments, locations of the devices can belinked with antigens (e.g., peptides, proteins, carbohydrates, lipids,proteoglycans, glycoproteins, etc.) in order to assay for antibodies ina bodily fluid sample by sandwich assay, competitive assay, or otherformats. Alternatively, the locations of the device may be addressedwith antibodies, in order to detect antigens in a sample by sandwichassay, competitive assay, or other assay formats. As the isoelectricpoint of antibodies and proteins can be determined fairly easily byexperimentation or pH/charge computations, the electronic addressing andelectronic concentration advantages of the devices may be utilized bysimply adjusting the pH of the buffer so that the addressed or analytespecies will be charged.

In some embodiments, the isolated nucleic acids are useful for use inimmunoassay-type arrays or nucleic acid arrays.

Electrode Arrays

In various embodiments, microelectrodes are arranged in an array. Theadvantages of microelectrode array deigns include increasing thegradient of an electric field generated while also reducing the ACelectrothermal flow generated at any particular voltage. In anembodiment, the microelectrode array comprises a floating electrode,i.e., an electrode surrounding the working electrode by not beingenergized during ACE. FIG. 12 shows an example of flow velocity profile(left) and a DEP gradient generated by the microelectrode array with analternating configuration of regular electrodes and floating electrodes.Table 1 shows the performance derived from different configurations ofmicroarray electrode arrays.

TABLE 1 Comparison of performance parameters for different floatingelectrode designs and basic design with floating electrodes. FloatingRing Max Max Gradient of total Electrode width E-field Velocity electricfield current Width (μm) (μm) (V/m) (m/s) (mKg²/s⁶A²) 2 × 2 (A) 5 107.313E+05 2.443E−05 6.408E+18 8.46E−04 5 12.5 7.139E+05 2.662E−054.686E+18 8.72E−04 5 15 7.133E+05 2.729E−05 5.587E+18 8.89E−04 5 17.57.053E+05 2.793E−05 5.122E+18 9.01E−04 5 20 6.960E+05 2.803E−054.655E+18 9.09E−04 5 N/A 7.018E+05 2.798E−05 5.511E+18 9.17E−04 Regular4.614E+05 4.044E−05 6.569E+17 9.03E−04

As can been seen in Table 1, there is one order of magnitude increase inthe gradient of electric field in comparison to the regular design,i.e., the microarray electrode array without a floating electrode.Employing floating electrodes in some embodiments, the DEP force(F_(DEP)) is greater or much great than the flow force (F_(FLOW)), thusallowing to use lower voltage to achieve capture. Based on the use offloating electrodes, systems or devices requiring low power consumptionwill be fabricated.

DEFINITIONS AND ABBREVIATIONS

The articles “a”, “an” and “the” are non-limiting. For example, “themethod” includes the broadest definition of the meaning of the phrase,which can be more than one method.

“Vp-p” is the peak-to-peak voltage.

“TBE” is a buffer solution containing a mixture of Tris base, boric acidand EDTA.

“TE” is a buffer solution containing a mixture of Tris base and EDTA.

“L-Histidine buffer” is a solution containing L-histidine.

“DEP” is an abbreviation for dielectrophoresis.

“ACE” is an abbreviation for Alternate Current Electrokinetics.

“ACET” is an abbreviation for AC electrothermal.

EXAMPLES Example 1

A two-chamber fluidics cartridge containing a hydrogel coatedmicrolectrode array was loaded into an ATS system. The microelectrodearray comprised electrodes in a hollow ring shape, as depicted in FIG.5. In one chamber, a standard solution with conductivity of 0.8 S/m andspiked DNA (genomic purchased from Promega or Lambda purchased fromBioLabs) at 25 pg/μL was loaded for a total volume of 530 μL. In theother chamber, an unknown sample in a bodily fluid (blood, serum,plasma, sputum, etc. . . . ) was loaded to a total of 530 μL. The DNAwas stained at a ratio of 1:5000× using YOYO®-1 green fluorescent dyepurchased from Life Technologies. Both liquids were run on the ATSsystem at 10 Volts peak-to-peak and 15 kHz for 10 minutes while flowingat a variable flow rate (5 to 250 μL/min) (FIGS. 6 and 7). The arrayswere then washed with an isotonic buffer (water+osmolites) for another10 minutes at a variable flow rate in order to remove all matter thatwas not captured on the electrodes. At the end of the 20 minute process,an image of the microelectrode array was taken (one in each chamber)using a CCD camera with a 10× objective on a microscope using greenfluorescent filters (FITC) (FIG. 8). This allowed for imagequantification of the captured matter of the unknown sample incomparison to the known sample. After the ACE power was turned off andthe captured matter was released from the microelectrode array (FIG. 9),the fluid into which the capture matter was released was retrieved fromthe cartridge and collected for subsequent analysis.

Example 2

Various electrode designs were tested according to the methods describedin Example 1. Generally, electrode geometry that increased F_(DEP) whileattenuating F_(FLOW) enabled the stronger capture of nanoscale analytes.Below is a description of ACE performance difference between electrodedesigns.

TABLE 2 Description of ACE performance differences between electrodedesigns. Electrode Design Remarks Hollow Disk Standard electrodegeometry as shown in FIGS. 1, 6, 7, 8 Hollow Ring Increased surface areafor nanoscale analyte capture. Modification of flow pattern. Shown inFIG 2. Wavy Line Provides larger surface area for nanoscale analytecapture. Generates uni-axial flow. Shown in FIGS. 3 & 4. Hollow ringwith Reduces the ACET and ACEO. Shown in FIG. 5. extruded center BlockedElectrode Reduces the ACET and ACEO. Not shown. Floating ElectrodeReduces ACET and ACEO, collectively F_(FLOW), while increasing F_(DEP).Shown in FIG. 12.

While preferred embodiments of the present invention have been shown anddescribed herein, it will be obvious to those skilled in the art thatsuch embodiments are provided by way of example only. Numerousvariations, changes, and substitutions will now occur to those skilledin the art without departing from the invention. It should be understoodthat various alternatives to the embodiments of the invention describedherein may be employed in practicing the invention. It is intended thatthe following claims define the scope of the invention and that methodsand structures within the scope of these claims and their equivalents becovered thereby.

What is claimed is:
 1. A device for isolating a nanoscale analyte in asample, the device comprising: a. a housing; and b. alternating current(AC) electrodes within the housing, wherein the AC electrodes areconfigured to be selectively energized to establish AC electrokinetichigh field and AC electrokinetic low field regions, and the ACelectrodes comprise conductive material within the AC electrodes forreducing, disrupting or altering fluid flow around or within thevicinity of the AC electrodes as compared to fluid flow in regionsbetween or substantially beyond the vicinity, wherein the conductivematerial is substantially absent from the center of the individual ACelectrodes and the AC electrodes are configured in three-dimensions. 2.The device of claim 1, wherein the individual AC electrodes areconfigured in a hollow ring shape.
 3. The device of claim 1, wherein theindividual AC electrodes are configured in a hollow tube shape.
 4. Thedevice of claim 1, wherein the AC electrodes further comprisenon-conductive material.
 5. The device of claim 4, wherein thenon-conductive material surrounds the conductive material within the ACelectrodes and serves as a physical barrier to the conductive material.6. The device of claim 4, wherein the conductive material within the ACelectrodes fills depressions in the non-conductive material.
 7. Thedevice of claim 1, wherein the conductive material of thethree-dimensional AC electrodes increases the total surface area of theconductive material within the AC electrodes.
 8. The device of claim 1,wherein the conductive material within the AC electrodes is configuredat an angle.
 9. The device of claim 1, wherein the conductive materialwithin the AC electrodes is configured into angles between neighboringplanar electrode surfaces of equal to or less than 180 degrees and equalto or more than 60 degrees.
 10. The device of claim 1, wherein theconductive material within the AC electrodes is configured into adepressed concave shape.
 11. The device of claim 1, wherein theindividual AC electrodes are 40 μm to 100 μm in diameter.
 12. The deviceof claim 1, wherein the AC electrodes are in non-circularconfigurations.
 13. The device of claim 12, wherein an orientation anglebetween the non-circular configurations is between 25 and 90 degrees.14. The device of claim 13, wherein the non-circular configurationscomprise a wavy line configuration or a repeating unit comprising ashape of a pair of dots connected by a linker.
 15. The device of claim14, wherein the linker tapers inward toward the midpoint between thepair of dots.
 16. The device of claim 15, wherein the diameters of thedots are the widest points along the length of the repeating unit. 17.The device of claim 16, wherein an edge to edge distance between aparallel set of repeating units is equidistant, or roughly equidistant.18. The device of claim 1, wherein the AC electrodes comprise one ormore floating electrodes.
 19. The device of claim 18, wherein thefloating electrodes are not energized to establish AC electrokineticregions.
 20. The device of claim 18, wherein a floating electrodesurrounds an energized electrode.
 21. The device of claim 18, whereinthe floating electrodes induce an electric field with a higher gradientthan an electric field induced by non-floating electrodes.
 22. A methodfor isolating a nanoscale analyte in a sample, the method comprising: a.applying the sample to a device, the device comprising an array ofelectrodes capable of establishing an AC electrokinetic field region theAC electrodes are configured to be selectively energized to establish ACelectrokinetic high field and AC electrokinetic low field regions, andthe AC electrodes comprise conductive material within the AC electrodesfor reducing, disrupting or altering fluid flow around or within thevicinity of the AC electrodes as compared to fluid flow in regionsbetween or substantially beyond the vicinity, wherein the conductivematerial is substantially absent from the center of the individual ACelectrodes and the AC electrodes are configured in three-dimensions; b.producing at least one AC electrokinetic field region, wherein the atleast one AC electrokinetic field region is a dielectrophoretic highfield region; and c. isolating the nanoscale analyte in thedielectrophoretic high field region.
 23. The method of claim 22, whereinthe conductive material is configured in an open disk shape, a hollowring shape, a hollow tube shape or combinations thereof.
 24. The methodof claim 22, wherein a reduction in conductive material within theelectrodes results in reduced fluid flow in and around surfaces of theelectrodes, leading to an increase in nanoscale analyte capture on thesurfaces.
 25. The method of claim 22, further comprising lysing cells onthe array, wherein the cells are lysed using a direct current, achemical lysing agent, an enzymatic lysing agent, heat, pressure, sonicenergy, or a combination thereof.
 26. The method of claim 22, whereinthe array of electrodes further comprises non-conductive material. 27.The method of claim 26, wherein the non-conductive material surroundsthe conductive material within the electrodes and serves as a physicalbarrier to the conductive material.
 28. The method of claim 22, whereinthe sample comprises a fluid.
 29. The method of claim 28, whereinconductivity of the fluid is greater than or equal to 100 mS/m.
 30. Themethod of claim 22, wherein the nanoscale analyte is a nucleic acid.